Targeted liposome nano-drug preparation for acute exacerbation of pulmonary fibrosis as well as preparation method and application of targeted liposome nano-drug preparation

Abstract

The invention discloses a targeted liposome nano-drug preparation for the acute exacerbation stage of pulmonary fibrosis and a preparation method and application thereof, and belongs to the technical field of biological medicines.The preparation method comprises the steps that RGD modified lipid is prepared, soybean phospholipid, cholesterol, DSPE-PEG2K-Cy5.5, the RGD modified lipid, nintedanib and dexamethasone are added into a solvent to be mixed, rotary evaporation is conducted to obtain a lipid membrane, and the lipid membrane is dried to obtain the targeted liposome nano-drug preparation for the acute exacerbation stage of pulmonary fibrosis; and adding the lipid membrane into a hydration solution containing hyaluronic acid for hydration, carrying out ultrasonic treatment under an ice bath, extruding through a polycarbonate filter membrane, and freeze-drying. According to the preparation method, targeting of lung activated myofibroblasts and injured alveolar epithelial cells is realized, so that the accumulation efficiency of the medicine in lung lesions is improved, local synergistic anti-fibrosis and anti-inflammatory effects are realized, and the clinical outcome of AE-IPF is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis and a preparation method and application thereof. BACKGROUND

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease with unknown etiology. Its main pathological features are alveolar epithelial cell damage, fibroblast activation and abnormal deposition of extracellular matrix (ECM), leading to gradual destruction of alveolar structure and loss of function. Because the disease is irreversible, the prognosis of IPF patients is very poor, with a median survival time of 2-3 years.

[0003] About 20% of IPF patients will experience acute exacerbation (AE) during the course of the disease, which is characterized by sudden onset of dyspnea, worsening hypoxemia, and new extensive ground-glass opacities or pulmonary consolidation. AE-IPF currently has no specific standard treatment regimen, and usually relies on systemic glucocorticoids and / or anti-fibrotic small molecule drugs such as Nintedan.b. However, these drugs have certain limitations whether used alone or in combination.

[0004] In recent years, the drug delivery strategies developed for IPF have mainly focused on systemic oral or injection delivery, which is difficult to achieve lesion-specific drug deposition, resulting in limited local efficacy and increased adverse reactions. On the other hand, with the development of nanotechnology, liposomes, polymers, nanocrystals and other drug-loaded nanoplatforms have been gradually applied to the research and development stage of the treatment of lung diseases. Some of these preparations improve the drug concentration in the target organ by pulmonary inhalation delivery, which has the potential to improve efficacy and reduce toxicity and side effects.

[0005] However, existing delivery systems still have problems such as insufficient targeting, unbalanced drug release, inability to achieve combination therapy or unclear treatment mechanism, especially in the high inflammatory and high fibrotic environment of acute exacerbation. Therefore, a new generation of drug delivery system that can achieve lung lesion target recognition, combined delivery of anti-inflammatory and anti-fibrotic drugs, enrichment in high expression pathological target sites (such as integrin and CD44), and clear mechanism needs to be developed to further improve the accuracy and effectiveness of acute IPF treatment. SUMMARY

[0006] The present application aims to provide a targeted drug delivery treatment system for acute exacerbation idiopathic pulmonary fibrosis (AE-IPF). By co-encapsulating anti-fibrotic drug Nintedan.b and anti-inflammatory drug Dexamethasone, and modifying the surface of the drug delivery carrier with integrin and CD44 double-target ligands, the activated pulmonary myofibroblasts and damaged alveolar epithelial cells in the lung are targeted, thereby improving the accumulation efficiency of the drug in the lung lesions, achieving local synergistic anti-fibrotic and anti-inflammatory effects, and improving the clinical outcome of AE-IPF.

[0007] As one aspect of the present application, the present application provides a preparation method of a targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis, comprising the following steps,

[0008] Step 1: preparing RGD modified lipids: dissolving DSPE-PEG2K-NHS in a solvent; dissolving c(RGDfK) peptide in a solvent, mixing the obtained solutions, and performing reaction, dialysis and drying to obtain DSPE-PEG2K-RGD;

[0009] Step 2: adding soybean phospholipid, cholesterol, DSPE-PEG2K-Cy5.5, the RGD modified lipids, Nintedan.b and Dexamethasone into a solvent for mixing, rotary evaporation to obtain a lipid film, adding the lipid film into a hydrated solution containing hyaluronic acid for hydration, ultrasonic treatment under ice bath, extruding through a polycarbonate filter membrane, and freeze-drying to obtain the targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis.

[0010] As a preferred solution of the preparation method of the targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis, in step 1, the DSPE-PEG2K-NHS is dissolved in a solvent; the c(RGDfK) peptide is dissolved in a solvent, and the obtained solutions are mixed.

[0011] The solvent comprises DMSO; the concentration of DSPE-PEG2K-NHS in the obtained solution is 20-25 mg / mL, and the concentration of the c(RGDfK) peptide is 20-25 mg / mL.

[0012] As a preferred solution of the preparation method of the targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis, in step 1, the reaction comprises reaction for 2-3 hours under light shielding and normal temperature adjustment.

[0013] As a preferred solution of the preparation method of the targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis, in step 1, the dialysis comprises dialysis with a 3.5 kDa dialysis bag; and the drying comprises freeze-drying.

[0014] As a preferred scheme of the preparation method of the targeted liposome nanopharmaceutical preparation for acute exacerbation of pulmonary fibrosis according to the present application: in step 2, the mass ratio of soybean phospholipid, cholesterol, DSPE-PEG2K-Cy5.5, the RGD modified lipid, nintedanib, dexamethasone is 62-63: 14-15: 8-9: 14-15: 5-6: 1-2.

[0015] As a preferred scheme of the preparation method of the targeted liposome nanopharmaceutical preparation for acute exacerbation of pulmonary fibrosis according to the present application: in step 2, the rotary evaporation is performed at a temperature of 40-45 DEG C; the hyaluronic acid includes low molecular weight hyaluronic acid, and the average molecular weight is 10-20 kDa.

[0016] As a preferred scheme of the preparation method of the targeted liposome nanopharmaceutical preparation for acute exacerbation of pulmonary fibrosis according to the present application: in step 2, the concentration of hyaluronic acid in the hydration solution is 0.5 mg / mL, the hydration temperature is 37 DEG C, and the hydration time is 30-40 min.

[0017] The present application has the following beneficial effects: the present application modifies RGD short peptides and hyaluronic acid (HA) on the surface of liposomes, respectively recognizes integrins (such as alpha v beta 3, alpha v beta 6) and CD44 which are highly expressed in AE-IPF, and actively targets activated myofibroblasts and damaged / transformed alveolar epithelial cells; in the in vivo imaging experiment, the aggregation efficiency of the double-modified liposomes (ND-RHL) in the pulmonary consolidation area is significantly better than that of the unmodified preparation (ND-L), the drug bioavailability is improved, and the exposure of non-target organs is reduced; and the lung lesion targeted delivery and deep tissue penetration are realized.

[0018] The present application first encapsulates nintedanib and dexamethasone in a targeted nanoparticle, and delivers them by pulmonary inhalation; compared with single drug use, the present application can more comprehensively control the inflammatory storm and fibroblast activation process of AE-IPF, form an "upstream anti-inflammatory + downstream anti-fibrosis" disease chain control; in an animal model, the combination scheme of the present application significantly improves body weight maintenance, survival rate and lung tissue structure, while the single drug has limited effect, which reflects the synergistic advantage of drug efficacy.

[0019] Compared with the traditional system administration (oral administration / intravenous injection), the ND-RHL adopts an aerosol inhalation preparation delivery method, can directly act on the lung target organ, improve the local drug efficacy concentration, and significantly reduce the systemic exposure; under the same dose, the liver and kidney function indicators of the mice in the ND-RHL group do not change significantly, indicating that the preparation system has good safety and clinical transformation prospect.

[0020] Transcriptome analysis showed that the formulation of this invention can simultaneously inhibit key signaling pathways such as PI3K-AKT-mTOR, Wnt / β-catenin and NF-KB-PPARγ that are abnormally activated in AE-IPF; it can effectively reverse fibrosis progression and disrupt EMT process, and is a high-potential multifunctional drug system with a clear mechanism and precise targeting.

[0021] The ND-RHL of this invention adopts mature thin-film hydration-ultrasonic emulsification-film extrusion technology, which can achieve controllable particle size in the range of 130-160nm, PDI<0.2, and Zeta potential<-30mV. The materials of this invention are widely available, the synthesis steps are clear, and it has the conditions for scale-up production. The lyophilized powder form is convenient for clinical storage and transportation, the formulation system is stable and highly reproducible, and it meets the conversion requirements under GMP conditions.

[0022] In summary, this invention achieves breakthroughs in drug delivery strategies, target identification, delivery methods, and mechanism interventions, enabling more effective solutions to the challenges in AE-IPF treatment. It demonstrates significant advantages in improving efficacy, enhancing safety, and promising prospects for industrial application, providing a novel approach for personalized and precise treatment of pulmonary fibrosis-related diseases. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0024] Figure 1 This is a schematic diagram of the assembly of ND-RHL nanoliposomes.

[0025] Figure 2 The figures show particle size, TEM, encapsulation / drug loading / modification efficiency, and drug synergy analysis of the control group ND-L and the experimental group ND-RHL.

[0026] Figure 3 Flow cytometry plot of RGD / CD44 targeting cell entry.

[0027] Figure 4 This is a micro-CT imaging image of the lungs.

[0028] Figure 5 This is a diagram illustrating the phenotypic analysis of fibrosis and inflammation.

[0029] Figure 6 This is a diagram illustrating the in vivo drug safety evaluation.

[0030] Figure 7 This diagram serves as a verification of the therapeutic mechanism. Detailed Implementation

[0031] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0032] Example 1:

[0033] This embodiment discloses a method for preparing a targeted liposome formulation ND-RHL by chemically coupling modified RGD short peptides with hyaluronic acid. This formulation is for intrapulmonary delivery and is used to treat acute exacerbation of idiopathic pulmonary fibrosis. The specific preparation method is as follows:

[0034] 1. Experimental materials:

[0035]

[0036]

[0037] The RGD peptide is a cyclic pentapeptide with the sequence Arg-Gly-Asp-D-Phe-Lys, which forms a closed ring structure through an amide bond between the carboxyl group of the aspartic (Asp) side chain and the ε-amino group of the lysine (Lys) side chain. The molecular formula of the cyclic peptide is C1. 30 H 48 N 10 O7, with a molecular weight of approximately 644.77 g / mol, is used for integrin receptor targeting.

[0038] 2. Synthesis of RGD-modified lipid DSPE-PEG2 K-RGD:

[0039] (1) Dissolve DSPE-PEG2 K-NHS (10 mg) completely in 250 μL of anhydrous DMSO;

[0040] (2) Dissolve c(RGDfK) peptide (10 mg) in 250 μL of anhydrous DMSO;

[0041] (3) Mix the solutions obtained in steps (1) and (2) and react them at room temperature in the dark for 2 hours (with gentle shaking) to complete the coupling;

[0042] (4) After obtaining the thioester bond-forming product in step (3), unreacted small molecules are removed by dialysis using a dialysis bag (MWCO 3.5kDa), and then freeze-dried to obtain powdered DSPE-PEG2 K-RGD for later use.

[0043] 3. Preparation of ND-RHL liposome formulations:

[0044] (1) The components are shown in Table 1:

[0045] Table 1

[0046]

[0047]

[0048] (2) Specific steps:

[0049] The lipids and drugs in Table 1 above are dissolved in a chloroform:methanol mixture of 4:1.

[0050] The lipid membrane was dried and homogeneous by rotary evaporation at 40°C.

[0051] The hydration solution is ultrapure water containing 0.5 mg / mL low molecular weight hyaluronic acid (10-20 kDa);

[0052] The lipid membrane was added to 10 mL of ultrapure water containing 0.5 mg / mL low molecular weight hyaluronic acid (10-20 kDa), hydrated at 37°C for 30 minutes, and then sonicated in an ice bath for 5 minutes to form liposomes.

[0053] ND-RHL liposomes with uniform particle size distribution were obtained by extruding through a 100nm polycarbonate filter membrane 10 times; 2 × lipid mass of sucrose was added, pre-frozen at -80℃, and freeze-dried for 48 hours to obtain ND-RHL freeze-dried powder.

[0054] Example 2:

[0055] Lung nebulization delivery and animal model validation:

[0056] 1. Establishment of animal models:

[0057] C57BL / 6 male mice (8 weeks old) were used to construct the AE-IPF model. The method is as follows:

[0058] Day 0: Intratracheal infusion of 3 mg / kg bleomycin (BLM);

[0059] Day 7 and Day 10: Administer an additional 0.5 mg / kg LPS to induce acute exacerbations;

[0060] Treatment began on Day 8.

[0061] 2. Administration method:

[0062] After reconstituted ND-RHL lyophilized powder, it is administered via a nebulizer. The dosage is calculated as follows:

[0063] Nintedanib: 2 mg / kg

[0064] Dexamethasone: 0.4 mg / kg

[0065] The medication was administered on Day 8, 11, 14, 17, and 21, for a total of 5 times.

[0066] 3. Key points for result evaluation:

[0067] micro-CT results: Ground-glass opacities in the lungs were significantly reduced in the treatment group;

[0068] H&E staining: Alveolar structure is well preserved, with no consolidation or enlargement;

[0069] MaSSon staining: Collagen content was significantly reduced;

[0070] The bronchoalveolar lavage fluid (BALF) showed a significant decrease in inflammatory factors (TNF-α, IL-6, and TGF-β1); the animals' body weight, Ashcroft score, and hydroxyproline levels were significantly better than those in the positive control group.

[0071] This indicates that the preparation has good lung targeting and therapeutic effect.

[0072] The experimental results are as follows:

[0073] Figure 1 This is a schematic diagram of the assembly of ND-RHL nanoliposomes. Figure 1 The lipid bilayer structure, RGD and HA modification sites, and dual-drug encapsulation positions of ND-RHL and the control group ND-L (without RGD and HA modification) are shown.

[0074] Figure 2 The figures show particle size, TEM images, encapsulation / drug loading / modification efficiency, and drug synergy analysis of the control group ND-L and the experimental group ND-RHL. Figure 2 In the image, (A) particle size of ND-L and ND-RHL; (B) potential of ND-L and ND-RHL; (C, D) stability of particle size and potential of ND-L and ND-RHL over time; (E) transmission electron microscopy (TEM) images of ND-L and ND-RHL; (F) statistics on particle size, potential, encapsulation (EE%) / drug loading (DL%) / modification efficiency (CE%) of ND-L and ND-RHL formulations. Figure 2 It can be seen that the particle size is concentrated in the range of 130-160 nm, and electron microscopy shows that the double membrane is clear, round and regular. A549(G) and MRC5(H) cells were treated with (G, H)N and D drugs, and the CI curve of synergistic effect was calculated. In both effector cells, the CI value was about 0.3-0.4, which was less than 1, indicating a strong synergistic effect.

[0075] The physicochemical properties of ND-RHL are shown in Table 2:

[0076] Table 2

[0077] Item Value (mean ± SD) Particle size 152.0 ± 5.1 nm Zeta potential -43.3 ± 1.0 mV EE% (Nintedanib) 83.3±1.1％ EE% (Dexamethasone) 86.5±0.5％ DL% (Nintedanib) 5.00±0.068％ DL% (Dexamethasone) 1.04±0.006％ CE% (RGD) 1.15±0.13％ CE% (HA) 3.44±0.25％

[0078] Figure 3 Flow cytometry plot of RGD / CD44 targeting cell entry. Figure 3In the diagram, (A) is a schematic diagram of cell overexpression of ITGAV / CD44 and liposome treatment; (BD) flow cytometry analysis of intracellular uptake signal intensity of ND-L and ND-RHL; (E, F) confocal microscopy detection of intracellular uptake signals of ND-L and ND-RHL, with red indicating cy5.5-labeled liposome signals, and a scale bar of 100 μm. Figure 3 It can be seen that the RGD+HA modified ND-RHL liposomes have significantly stronger cellular uptake than the control group, exhibiting significant intracellular uptake selectivity.

[0079] Figure 4 This is a micro-CT imaging image of the lungs. Figure 4 The image shows: a schematic diagram of the modeling grouping (A) and process (B); micro-CT and 3D reconstructed images (C); and statistical charts of poorly ventilated areas (D) and lung parenchyma volume (E). Figure 4 It can be seen that the area of ​​poor lung ventilation was significantly reduced after ND-RHL treatment.

[0080] Figure 5 This is a diagram illustrating the phenotypic analysis of fibrosis and inflammation. Figure 5 In the image, (A) HE and Masson staining images, scale bar = 1 mm, 100 μm; Ashcroft score (B), fibrosis area (C), and hydroxyproline content statistics (D); (EH) bronchoalveolar lavage fluid (BALF) inflammatory factor levels. Figure 5 It can be seen that after ND-RHL treatment, the alveolar tissue structure is restored, and the levels of fibrosis and inflammation are reduced.

[0081] Figure 6 This is a diagram illustrating the in vivo drug safety evaluation. Figure 6 In the image, (A) HE staining of major organs, scale bar = 100 μm; (B) mouse weight gain curve; (CF) serum markers of liver (ALT, AST) and kidney (CUN, CRE) function. Figure 6 It can be seen that there is no organ damage or drug toxicity after ND-RHL treatment.

[0082] Figure 7 This diagram serves as a verification of the therapeutic mechanism. Figure 7 In the image, (A) is a schematic diagram of RNA-seq; (B, C) are GO clustering heatmaps (B) and KEGG bubble diagrams (C); (D) is a schematic diagram of lung tissue signaling pathway-related protein analysis; and (EH) are Western blot plots of proteins related to the EMT, PI3K-AKT-mTOR, Wnt / β-catenin, and NF-κB-PPARγ signaling axes. Figure 7 It can be seen that ND-RHL exerts its therapeutic effect by inhibiting the PI3K-AKT-mTOR, Wnt / β-catenin, and NF-κB-PPARγ signaling axes.

[0083] In summary, in this invention, the RGD ligand specifically recognizes integrins (such as αvβ6 and αvβ3), and HA recognizes the CD44 receptor; both are highly expressed in AE-IPF lesions. The liposome particle size is suitable for airway delivery, allowing for local release of the active drug. Nintedanib, in combination with dexamethasone, activates myofibroblasts and inflammatory cell populations, exerting anti-fibrotic and anti-inflammatory effects. The formulation of this invention is inhaled via an ultrasonic nebulizer, once every 2-3 days, with the most significant effect achieved after 5 consecutive inhalations. The formulation exhibits good stability, remaining stable for more than 7 days at 4°C, and its shelf life can be extended to 12 months through freeze-drying.

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a targeted liposomal nanomedicine formulation for acute exacerbations of pulmonary fibrosis, characterized in that: Includes the following steps, Step 1: Preparation of RGD modified lipids: Dissolve DSPE-PEG2K-NHS in a solvent; dissolve c(RGDfK) peptide in a solvent, mix the resulting solutions, react, dialyze, and dry to obtain DSPE-PEG2K-RGD; Step 2: Soybean lecithin, cholesterol, DSPE-PEG2K-Cy5.5, the RGD modified lipid, nintedanib, and dexamethasone are added to a solvent and mixed. The mixture is then rotary evaporated to obtain a lipid membrane. The lipid membrane is added to an aqueous solution containing hyaluronic acid for hydration, sonicated under ice bath conditions, extruded through a polycarbonate filter membrane, and freeze-dried to obtain the targeted liposome nanomedicine formulation for acute exacerbations of pulmonary fibrosis.

2. The method for preparing a targeted liposome nanomedicine formulation for acute exacerbation of pulmonary fibrosis according to claim 1, characterized in that: In step 1, DSPE-PEG2K-NHS is dissolved in a solvent; c(RGDfK) peptide is dissolved in a solvent, and the resulting solutions are mixed. The solvent includes DMSO; the concentration of DSPE-PEG2K-NHS in the obtained solution is 20-25 mg / mL, and the concentration of c(RGDfK) peptide is 20-25 mg / mL.

3. The method for preparing a targeted liposomal nanomedicine formulation for acute exacerbation of pulmonary fibrosis according to claim 1 or 2, characterized in that: In step 1, the reaction is carried out, including reacting for 2 to 3 hours under light-protected and room-temperature conditions.

4. The method for preparing a targeted liposome nanomedicine formulation for acute exacerbation of pulmonary fibrosis according to claim 1 or 2, characterized in that: In step 1, the dialysis includes dialysis using a 3.5 kDa dialysis bag; the drying includes freeze drying.

5. The method for preparing a targeted liposomal nanomedicine formulation for acute exacerbation of pulmonary fibrosis according to claim 1 or 2, characterized in that: In step 2, the mass ratio of soybean phospholipids, cholesterol, DSPE-PEG2K-Cy5.5, the RGD modified lipids, nintedanib, and dexamethasone is 62-63:14-15:8-9:14-15:5-6:1-2.

6. The method for preparing a targeted liposome nanomedicine formulation for acute exacerbation of pulmonary fibrosis according to claim 1 or 2, characterized in that: In step 2, the rotary evaporation is carried out at a temperature of 40-45°C; the hyaluronic acid includes low molecular weight hyaluronic acid with an average molecular weight of 10-20 kDa.

7. The method for preparing a targeted liposomal nanomedicine formulation for acute exacerbation of pulmonary fibrosis according to claim 1 or 2, characterized in that: In step 2, the concentration of hyaluronic acid in the hydration solution is 0.5 mg / mL, the hydration temperature is 37℃, and the hydration time is 30–40 min.

8. The liposome nanomedicine preparation obtained by the preparation method of the targeted liposome nanomedicine preparation for acute exacerbation of pulmonary fibrosis according to claim 1.

9. The use of the liposome nanomedicine formulation according to claim 8 in the preparation of a drug formulation for treating acute exacerbations of pulmonary fibrosis.

10. The application according to claim 9, characterized in that: The pharmaceutical preparations include those for nebulization.

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