Composite drug-loaded nano preparation and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a composite drug-loaded nano preparation and a preparation method thereof. The composite drug-loaded nano-preparation comprises a drug-loaded nano-liposome, and a biological enzyme and a myofibroblast targeting ligand which are loaded on the surface of the drug-loaded nano-liposome, the drug-loaded nano-liposome is a nano-liposome in which an anti-fibrosis drug is encapsulated; the mass ratio of the drug-loaded nano-liposome to the biological enzyme to the myofibroblast targeting ligand is (6-15): (0.5-4): (0-3). According to the invention, a composite drug-loaded nano preparation for pulmonary drug delivery in an aerosol inhalation manner is constructed, and the myofibroblast targeting ligand and the biological enzyme are jointly modified on the surface of the drug-loaded nano liposome; and the composite nano preparation (CLG-CS-FAP-NIN) which has myofibroblast targeting and can degrade excessive collagen in an extracellular matrix is prepared and is used for treating the pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composite drug-loaded nano preparation and a preparation method thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease of unknown etiology. It is progressive and ultimately fatal. It is a life-threatening lung disease. There is still no clear cure, and treatment options are limited. There are only two relatively effective drugs that cannot improve the overall survival rate of patients. Therefore, the 5-year survival rate is less than 30%, and it is called a "tumor-like disease." In recent years, nanoparticle aerosol inhalation can deliver drugs directly to the lesion site, improve drug utilization and reduce systemic adverse reactions, and has become a promising treatment strategy. However, despite the good prospects of aerosol inhalation of nanoparticles for the treatment of pulmonary fibrosis, there are still many difficulties and challenges.

[0003] In patients with pulmonary fibrosis, due to continuous inflammatory stimulation, imbalance of repair mechanisms, and abnormal release of multiple growth factors, a large amount of collagen accumulates in the lungs, which not only compresses the normal alveolar structure, but also forms a physical barrier, making it difficult for therapeutic drugs to enter the diseased area. In addition to increased collagen deposition, the structure of excessively deposited collagen has also changed. Under physiological conditions, collagen fibers have a certain arrangement that can provide the elasticity and flexibility required by lung tissue. In the environment of pulmonary fibrosis, the arrangement of collagen fibers becomes more disordered and irregular, forming a denser and harder barrier. This structural change makes it more difficult to deliver nanoparticles, further limiting their delivery and distribution in areas of pulmonary fibrosis.

[0004] Pulmonary fibrosis is a heterogeneous lung disease involving multiple pathological mechanisms and different histopathological changes. However, most current nanoparticles lack targeted modifications in their design, which means that they cannot effectively recognize and concentrate around diseased tissues in vivo. Therefore, although atomization can deliver drugs directly to the lungs, its targeting is still insufficient, especially for specific lesion areas. The atomized inhaled drugs may not be concentrated in the specific lesions and are often absorbed or excreted before the drugs are concentrated at the target, resulting in uneven drug distribution, thereby reducing the therapeutic effect.

[0005] Since the pathogenesis of pulmonary fibrosis is very complex, the pathology, clinical manifestations and prognosis are all different, and the therapeutic effect of only targeting one pathogenesis is limited, so the current treatment of pulmonary fibrosis needs to consider multi-drug combination and synergistic treatment in order to achieve better treatment effects for pulmonary fibrosis. With the continuous development and application of inhalable nanoparticles, the selection of appropriate anti-fibrotic drugs and the construction and optimization of nanoprobe drug delivery systems are crucial to achieve precise targeting of fibrotic diseased sites and the development and design of nanoformulations that can be aerosolized and inhaled and can achieve better therapeutic effects. Summary of the invention

[0006] Based on the above content, the present invention provides a composite drug-loaded nanoformulation and a preparation method thereof. The composite drug-loaded nanoformulation of the present invention can be non-invasively targeted and delivered to the site of pulmonary fibrosis by atomization inhalation, thereby improving the utilization rate of the drug. The composite drug-loaded nanoformulation of the present invention has a small dosage, can effectively improve the efficacy of pulmonary fibrosis, and has higher safety.

[0007] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is a composite drug-loaded nanoformulation, comprising a drug-loaded nanoliposome, and a biological enzyme and a myofibroblast targeting ligand loaded on the surface of the drug-loaded nanoliposome; The drug-loaded nanoliposomes are nanoliposomes encapsulating anti-fibrosis drugs; The mass ratio of the drug-loaded nanoliposome to the biological enzyme and myofibroblast targeting ligand is (6-15): (0.5-4): (0-3).

[0008] The second technical solution of the present invention is a method for preparing the above-mentioned composite drug-loaded nanoformulation, comprising the following steps: The drug-loaded nanoliposomes and the myofibroblast targeting ligand were reacted under nitrogen sealing and dialyzed to obtain nanoformulation A; Adding the nano preparation A to the chitosan solution for reaction and dialyzing to obtain the nano preparation B; The nano preparation B is reacted with a biological enzyme and dialyzed to obtain the composite drug-loaded nano preparation.

[0009] The present invention discloses the following technical effects: (1) The present invention provides a composite drug-loaded nanoformulation that can be administered to the lungs by aerosol inhalation. Compared with oral or intravenous administration, aerosol inhalation can effectively deliver drugs to the lung area, improve the local targeted therapeutic effect of drugs, increase the bioavailability of drugs, reduce the risk of systemic side effects, and reduce patients' rejection of drugs, thereby improving treatment compliance.

[0010] (2) The composite drug-loaded nanoformulation provided by the present invention flexibly combines multiple components such as biological enzymes (collagenase), myofibroblast targeting ligands (FAP targeting peptides) and anti-fibrotic drugs (nintedanib) together, and forms a multi-effect integrated treatment system through atomization inhalation treatment, which can comprehensively treat different pathological mechanisms of pulmonary fibrosis, thereby producing better therapeutic effects.

[0011] (3) The biological enzyme (collagenase) in the composite drug-loaded nanoformulation provided by the present invention plays a role in collagen disintegration, eliminates excessive collagen deposition, reduces fibrotic scar tissue, helps to restore alveolar structure and improve lung function, breaks through a major barrier in the treatment of pulmonary fibrosis, and helps deliver drugs to deep lesions.

[0012] (4) The myofibroblast targeting ligand (FAP targeting peptide) in the composite drug-loaded nanoformulation provided by the present invention enables the nanoformulation to more directly and accurately target the activated myofibroblasts in the lesion site after breaking through the collagen barrier, thereby increasing the enrichment of the drug in fibrotic tissue, solving the problem of poor targeting of conventional drugs and reducing the impact on healthy tissues.

[0013] (5) Nintedanib in the composite drug-loaded nanoformulation provided by the present invention can ultimately bind precisely to the receptors on the myofibroblasts in the lesion site through the previous collagen penetration and FAP targeting, exerting anti-inflammatory and anti-fibrotic effects, further inhibiting the fibrosis process, achieving precise treatment of pulmonary fibrosis lesions, achieving a more comprehensive and effective treatment effect, and providing a new strategy for the treatment of pulmonary fibrosis.

[0014] (6) The composite drug-loaded nanoformulation provided by the present invention uses 10% sucrose solution as the hydration liquid, and then modifies the surface of the drug-loaded nanoliposome by utilizing the electrostatic binding effect of chitosan and collagenase, which can improve the connection rate of collagenase and the stability of the nanoformulation, prevent the aggregation or deposition of particles, and make the nanoformulation still have good stability after atomization shearing, which is conducive to long-term storage. It also has the advantages of small particle size, good biocompatibility, sustained drug release, simple synthesis process, mild reaction conditions, etc., which is conducive to synthesis scale-up and clinical transformation, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1This is a liquid appearance diagram of the composite drug-loaded nanoformulation (abbreviated as: nanoformulation) prepared in Examples 1-4 of the present invention.

[0017] Figure 2 The particle size diagram and potential diagram of the composite drug-loaded nanoformulation prepared in Examples 1-4 of the present invention; wherein (a) is the particle size diagram, and (b) is the potential diagram.

[0018] Figure 3 This is a Coomassie Brilliant Blue SDS-PAGE protein staining image of the composite drug-loaded nanoformulation prepared in Examples 1-4 of the present invention.

[0019] Figure 4 The graphs are comparative diagrams of the particle size change and zeta potential change of the composite drug-loaded nanoformulations prepared in Examples 1-4 of the present invention before and after atomization; wherein (a) is the particle size change and (b) is the zeta potential change.

[0020] Figure 5 The graphs show the in vitro stability test results and the in vitro sustained release curves under different conditions of the composite drug-loaded nanoformulation (CLG-CS-FAP@NIN) prepared in Example 4 of the present invention; (a) is the in vitro stability test result graph, and (b) is the in vitro sustained release curves under different conditions.

[0021] Figure 6 The cytotoxicity of the composite drug-loaded nanoformulations prepared in Examples 1-4 of the present invention on lung cells (BEAS-2B, RAW264.7, NIH-3T3, and NIH-3T3 induced by TGF-β); wherein (a) is BEAS-2B, (b) is RAW264.7, (c) is NIH-3T3, and (d) is NIH-3T3 induced by TGF-β.

[0022] Figure 7 The graphs show the results of cellular uptake of the composite drug-loaded nanoformulations prepared in Examples 1-4 of the present invention in NIH-3T3 and NIH-3T3 induced by TGF-β; wherein (a) is NIH-3T3, and (b) is NIH-3T3 induced by TGF-β.

[0023] Figure 8 HE, Masson and Sirius red staining images of lung tissues of healthy mice and bleomycin-induced pulmonary fibrosis mouse model of the present invention.

[0024] Fig. 9 The survival curve statistics and body weight change statistics of each group of mice after aerosol inhalation treatment of the composite drug-loaded nanoformulation prepared in Examples 1-4 of the present invention; wherein (a) is a survival curve statistics, and (b) is a body weight change statistics.

[0025] Fig.10This is a picture showing the appearance of lung tissues of mice in each group after aerosol inhalation treatment of the composite drug-loaded nanoformulation prepared in Examples 1-4 of the present invention.

[0026] Fig.11 The composite drug-loaded nanoformulations prepared in Examples 1-4 of the present invention are statistical graphs of lung hydroxyproline content and total cell counts in bronchoalveolar lavage fluid of each group of mice after aerosol inhalation treatment; wherein (a) is a statistical graph of lung hydroxyproline content, and (b) is a graph of total cell counts in bronchoalveolar lavage fluid.

[0027] Fig.12 HE staining images of various tissues (heart, liver, spleen, and kidney) of healthy mice after aerosol inhalation of the composite drug-loaded nanoformulation prepared in Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The "%" described in the present invention, unless otherwise specified, refers to mass percentage.

[0034] In order to solve the problems of low drug utilization, poor targeting and poor stability of existing nano-preparations, an efficient, targeted and non-invasive treatment strategy is provided for the treatment of pulmonary fibrosis. The nano-preparation involved in the present invention is based on nano-liposomes encapsulating anti-fibrotic drugs, and biological enzymes and myofibroblast targeting ligands (collagenase and FAP targeting peptide) are connected on the surface of the liposomes. First, the nano-preparation is accurately delivered to the lungs by atomization inhalation, and then collagenase is used to degrade excessively deposited collagen in the extracellular matrix, so that the FAP targeting peptide targets myofibroblasts with high FAP expression, so that the anti-fibrotic drug (nintedanib) is accurately targeted to play a role in treating pulmonary fibrosis, reducing the dosage and adverse reactions of the drug, and reducing the collagen deposited in the pulmonary interstitium and inhibiting the production of collagen, reducing the severity of pulmonary fibrosis, and treating pulmonary fibrosis through a multi-effect treatment system to improve lung function.

[0035] The first aspect of the present invention provides a composite drug-loaded nanoformulation, comprising a drug-loaded nanoliposome, and a biological enzyme and a myofibroblast targeting ligand loaded on the surface of the drug-loaded nanoliposome; The drug-loaded nanoliposomes are nanoliposomes encapsulating anti-fibrosis drugs; The mass of the drug-loaded nanoliposome, the biological enzyme and the myofibroblast targeting ligand is (6-15): (0.5-4): (0-3).

[0036] In the present invention, the biological enzyme plays a role in degrading the extracellular matrix barrier; the myofibroblast targeting ligand plays a role in targeting myofibroblasts.

[0037] In a preferred embodiment of the present invention, the drug-loaded nanoliposomes include phospholipids, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG 2000 -Mal) and anti-fibrotic drugs by mass ratio (10~25): (0~3): (0.5~5): (0.5~4); The phospholipid is at least one of egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, and distearoylphosphatidylethanolamine-polyethylene glycol.

[0038] In a further preferred embodiment of the present invention, the phospholipid is dipalmitoylphosphatidylcholine.

[0039] In a preferred embodiment of the present invention, the drug-loaded nanoliposomes are prepared by thin film hydration method, ethanol injection method, ammonium sulfate gradient method, reverse evaporation method, freeze drying method or ultrasonic dispersion method.

[0040] Taking the thin film hydration method as an example, the preparation method of the drug-loaded nanoliposomes comprises the following steps: Phospholipids, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG 2000 -Mal) and anti-fibrotic drugs are dissolved in an organic solvent and then rotary evaporated and dried to obtain a film; Adding a hydrating liquid to the film, hydrating it in a water bath with ultrasonic wave and then performing ice bath with probe ultrasonic wave to obtain a suspension; The suspension was co-extruded through a 200 nm polycarbonate membrane to make its particle size distribution uniform, thereby preparing a nintedanib-containing nanoformulation containing Mal; The Mal-containing nano preparation carrying nintedanib is added to the chitosan solution and stirred for reaction under nitrogen sealing to obtain the drug-loaded nano liposome.

[0041] The organic solvent is at least one of chloroform, anhydrous ethanol, methanol and dimethyl sulfoxide.

[0042] The hydration liquid is one or more of deionized water, dripping water, sterilized sodium chloride solution, phosphate buffer, 10% glucose solution, and 10% sucrose solution, preferably 10% sucrose solution. The temperature of the water bath ultrasonic hydration is 40-45°C, and the time is 5-15 minutes.

[0043] The thin film hydration method is to perform hydration at a ratio of 10-30 mg of drug-loaded nanoliposomes to 1 ml of hydration solution.

[0044] In a preferred embodiment of the present invention, the anti-fibrosis drug is at least one of nintedanib, pirfenidone, metformin, resveratrol, curcumin, and trametinib.

[0045] In a further preferred embodiment of the present invention, the anti-fibrosis drug is nintedanib.

[0046] In a preferred embodiment of the present invention, the biological enzyme is collagenase.

[0047] The collagenase is at least one of type I collagenase, type II collagenase, type III collagenase and type IV collagenase.

[0048] In a further preferred embodiment of the present invention, the collagenase is type I collagenase.

[0049] In a preferred embodiment of the present invention, the myofibroblast targeting ligand is a FAP targeting peptide.

[0050] In some specific embodiments of the present invention, the average particle size of the composite drug-loaded nanoformulation is 127.07±1.56 nm, the zeta potential is -14.17±0.12 mV, and the encapsulation efficiency is 91.16±0.03%.

[0051] The second aspect of the present invention provides a method for preparing the above-mentioned composite drug-loaded nanoformulation, comprising the following steps: The drug-loaded nanoliposomes and the myofibroblast targeting ligands are reacted under nitrogen sealing, and dialyzed to obtain nanoformulation A; (this step allows the maleimide on the surface of the drug-loaded nanoliposomes to undergo Michael addition reaction with the thiol on the myofibroblast targeting ligands to form a drug-loaded nanoformulation loaded with the myofibroblast targeting ligands); Adding the nano preparation A to the chitosan solution for reaction and dialyzing to obtain the nano preparation B; The nano preparation B is reacted with a biological enzyme and dialyzed to obtain the composite drug-loaded nano preparation.

[0052] When preparing nanoformulation A, drug-loaded nanoliposomes and myofibroblast targeting ligands were magnetically stirred at 4° C. overnight under nitrogen sealing conditions.

[0053] When preparing the nanoformulation B, the nanoformulation A is added to the chitosan solution and the reaction conditions are as follows: the reaction is carried out under nitrogen atmosphere and magnetic stirring at room temperature for 1 to 4 hours.

[0054] When preparing the composite drug-loaded nanoformulation, the nanoformulation B and the biological enzyme are reacted under magnetic stirring at room temperature under nitrogen sealing for 1 to 4 hours.

[0055] In a preferred embodiment of the present invention, the concentration of chitosan in the chitosan solution is 0.1-2 mg / mL; more preferably, it is 0.1-0.5 mg / mL.

[0056] The solvent of the chitosan solution is at least one of dilute acid solutions such as hydrochloric acid, acetic acid, lactic acid, benzoic acid, and formic acid.

[0057] In a further preferred embodiment of the present invention, the solvent of the chitosan solution is an acetic acid solution with a volume concentration of 0.5-5%.

[0058] In order to overcome the collagen barrier, the present invention performs surface modification on the nanoparticles to improve their penetration ability. Collagenase, as a collagen hydrolase, can effectively decompose excess collagen in fibrotic tissue and fundamentally improve the pathological state of pulmonary fibrosis.

[0059] In order to improve the centralized targeting of aerosol inhaled drugs in pulmonary fibrosis lesions, the present invention adds targeted modification in the design process of nanoparticles, which helps to improve the therapeutic effect of the drugs. The present invention uses fibroblast activation protein targeting peptides to enable nanoparticles to specifically recognize and target myofibroblasts in the pulmonary fibrosis area, thereby directly acting on the lesion tissue and improving the therapeutic effect.

[0060] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0061] The collagenase used in the examples of the present invention was purchased from Beijing Solebow Technology Co., Ltd., originated from Clostridium histolyticum, and is type I collagenase; the enzyme activity is ≥125 units / mg.

[0062] The FAP targeting peptide used in the examples of the present invention was purchased from Anhui Zhuanpeptide Biotechnology Co., Ltd.

[0063] BEAS-2B, RAW264.7, and NIH-3T3 used in the present invention are from the American Type Culture Collection (ATCC).

[0064] The chitosan used in the embodiments of the present invention is deacetylated chitosan, and the degree of deacetylation is greater than 95%.

[0065] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] Example 1

[0067] This embodiment provides a method for preparing a nintedanib-loaded nanoformulation (CS@NIN), and the steps are as follows: S1: Accurately weigh 48 mg of dipalmitoylphosphatidylcholine (DPPC), 3.3 mg of cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG 2000 -Mal) 5.4 mg and nintedanib 3.6 mg were dissolved in chloroform (the amount of chloroform was sufficient to fully dissolve and mix the raw materials), and the mixture was evaporated under reduced pressure at 45°C and 120 rpm until a uniform film was formed, and then vacuum dried for 10 h to prepare a dry film; S2: Add 3 mL of 10% sucrose solution to the film prepared in S1, and hydrate it in a water bath with ultrasonic waves at 45°C for 10 min (power 150 W, time 10 min), and then perform ice bath probe ultrasonic waves (power 150 W, time 6 min) to obtain a suspension; S3: The suspension of S2 was extruded through a polycarbonate membrane with a pore size of 200 nm using a liposome extruder for multiple times, so that the suspension repeatedly passed through the polycarbonate membrane with a pore size of 200 nm to form a nintedanib-containing Mal nanoformulation (DPPC-NIN) of uniform size, and the excess nintedanib was removed by dialysis, and the nanoformulation was stored in a refrigerator at 4°C for future use; S4: dissolving chitosan in an acetic acid solution having a volume concentration of 1% to prepare a chitosan solution having a concentration of 0.2 mg / mL; S5: The Mal-containing nanoformulation (DPPC-NIN) loaded with nintedanib prepared in S3 and the chitosan solution in S4 were reacted at a volume ratio of 1:1 under magnetic stirring at room temperature for 2 h under nitrogen sealing to prepare a positively charged nanoformulation (CS@NIN) loaded with nintedanib. Excess chitosan was removed by dialysis and the nanoformulation was stored in a 4° refrigerator for later use.

[0068] Example 2

[0069] This embodiment provides a method for preparing a composite drug-loaded nanoformulation (CLG-CS@NIN) in which collagenase is loaded on the surface of drug-loaded nanoliposomes, and the steps are as follows: S1: The positively charged nintedanib nanoformulation (CS@NIN) prepared in Example 1 and the collagenase solution (10 mg / mL) were reacted at a volume ratio of 10:1 under nitrogen sealing and magnetic stirring at room temperature for 2 h to prepare the collagenase-loaded nintedanib nanoformulation (CLG-CS@NIN). The excess collagenase was removed by dialysis and stored in a refrigerator at 4°C for later use.

[0070] Example 3

[0071] This embodiment provides a method for preparing a composite drug-loaded nanoformulation (CS-FAP@NIN) in which a FAP targeting peptide is loaded on the surface of drug-loaded nanoliposomes, and the steps are as follows: S1: dissolving chitosan in acetic acid solution with a volume concentration of 1% to prepare a chitosan solution with a concentration of 0.2 mg / mL; S2: The Mal-containing nanoformulation (DPPC-NIN) loaded with nintedanib prepared in Example 1 and the FAP targeting peptide solution (20 mg / mL) were reacted at a volume ratio of 20:1 under nitrogen sealing at 4°C with magnetic stirring overnight to prepare the nintedanib nanoformulation loaded with FAP targeting peptide (FAP@NIN), and the excess FAP targeting peptide and nintedanib were removed by dialysis, and the nanoformulation was stored in a refrigerator at 4°C for future use; S3: The FAP targeting peptide-loaded nintedanib nanoformulation (FAP@NIN) prepared in S2 was mixed with the chitosan solution in S1 in a volume ratio of 1:1 and reacted with magnetic stirring at room temperature for 2 h under nitrogen sealing to obtain a positively charged FAP targeting peptide-loaded nintedanib nanoformulation (CS-FAP@NIN). Excess chitosan was removed by dialysis and the mixture was stored in a 4°C refrigerator for later use.

[0072] Example 4

[0073] This embodiment provides a method for preparing a composite drug-loaded nanoformulation (CLG-CS-FAP@NIN) in which collagenase and FAP targeting peptide are loaded on the surface of drug-loaded nanoliposomes, and the steps are as follows: S1: dissolving chitosan in acetic acid solution with a volume concentration of 1% to prepare a chitosan solution with a concentration of 0.2 mg / mL; S2: The Mal-containing nanoformulation (DPPC-NIN) loaded with nintedanib prepared in Example 1 and the FAP targeting peptide solution (20 mg / mL) were reacted at a volume ratio of 20:1 under nitrogen sealing at 4°C with magnetic stirring overnight to prepare the nintedanib nanoformulation loaded with FAP targeting peptide (FAP@NIN), and the excess FAP targeting peptide and nintedanib were removed by dialysis, and the nanoformulation was stored in a refrigerator at 4°C for future use; S3: The FAP targeting peptide-loaded nintedanib nanoformulation (FAP@NIN) prepared in S2 and the chitosan solution in S1 were reacted at a volume ratio of 1:1 under nitrogen atmosphere with magnetic stirring at room temperature for 2 h to prepare a positively charged FAP targeting peptide-loaded nintedanib nanoformulation (CS-FAP@NIN), and the excess chitosan was removed by dialysis; S4: The positively charged FAP targeting peptide-loaded nintedanib nanoformulation (CS-FAP@NIN) prepared in S3 was mixed with collagenase solution (10 mg / mL) at a volume ratio of 10:1 and reacted with magnetic stirring at room temperature for 2 h under nitrogen sealing to prepare the collagenase and FAP targeting peptide-loaded nintedanib nanoformulation (CLG-CS-FAP@NIN). The excess collagenase was removed by dialysis and the nanoformulation was stored in a 4°C refrigerator for later use.

[0074] The liquid appearance of the nanoformulations synthesized in Examples 1, 2, 3 and 4 is as follows: Figure 1 As shown, there is no precipitation and no stratification, which indicates that the present invention successfully prepares a nano-preparation in a good and stable state.

[0075] Effect Example 1 This effect example performs DLS characterization, encapsulation efficiency determination and other related characterization on the CS@NIN, CLG-CS@NIN, CS-FAP@NIN, and CLG-CS-FAP@NIN nanoformulations prepared in the above Examples 1-4: The above nanoformulations were tested by dynamic light scattering (DLS) to determine the particle size and zeta potential change, and the encapsulation efficiency of nintedanib was tested by high performance liquid chromatography (HPLC). The results are as follows: Figure 2 As shown in Table 1, it can be seen that the particle size of the nanopreparation prepared by the present invention is within 200 nm, and the nanopreparation maintains good stability; the encapsulation rate of nintedanib can reach more than 85%, with a good drug loading rate.

[0076] Table 1 , The above nanoformulation was tested by Coomassie Brilliant Blue SDS-PAGE protein staining to verify whether the collagenase was successfully connected. Figure 3 The results showed that CLG-CS@NIN and CLG-CS-FAP@NIN had obvious corresponding collagenase bands, indicating that collagenase was successfully connected to the nanoliposomes and CLG-CS@NIN and CLG-CS-FAP@NIN nanoformulations were successfully prepared.

[0077] Effect Example 2 This effect example performs DLS characterization and encapsulation efficiency determination on the CS@NIN, CLG-CS@NIN, CS-FAP@NIN, and CLG-CS-FAP@NIN nanoformulations prepared in Examples 1-4 before and after atomization: The results are as follows Figure 4 As shown in (a), (b) and Tables 2 and 3, it can be seen that the particle size and zeta potential of each nanoformulation after atomization only slightly increase, and the encapsulation efficiency is basically the same. This shows that the nanoformulation prepared by the present invention also has good physical and chemical properties after atomization, which is basically the same as before atomization, and can more accurately reflect the in vivo therapeutic effect of the nanoformulation in the present invention in animal experiments.

[0078] Table 2 , Table 3 .

[0079] Effect Example 3 This effect example tests the in vitro stability and in vitro sustained release effect of the CS@NIN, CLG-CS@NIN, CS-FAP@NIN, and CLG-CS-FAP@NIN nanoformulations prepared in Examples 1-4: The stability of the nanoformulation was tested by dynamic light scattering, and the particle size change of the nanoformulation was tested under different temperature conditions. The in vitro release of the nanoformulation was determined by the dialysis bag method. The in vitro sustained release experiment was carried out by measuring the sustained release of the drug in different release media (lung fluid, pH=6.8 PBS, pH=7.4 PBS) under the condition of a constant temperature shaker at 37°C.

[0080] Samples were taken at 4°C, 25°C and 37°C on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days, respectively, to measure the changes in the particle size of the nanoformulation; an appropriate amount of CLG-CS-FAP@NIN nanoformulation was placed in a dialysis bag (MWCO=3.5kd), and then the dialysis bag was placed in a container containing different release media. At 100rpm and 37°C, 600μL of release medium was taken out from the release medium at 1h, 2h, 4h, 8h, 12h, 24h, 48h and 72h for drug content detection, and then 600μL of the corresponding release medium was added. The sustained release of the drug at different time points was determined by high performance liquid chromatography, and the in vitro sustained release curve of the drug was drawn. The results are shown in Figure 2. Figure 5 As shown, (a) shows the stability at different temperatures, and (b) shows the drug release efficiency in different release media (lung fluid (corresponding to CLG-CS-FAP@NIN in BALF in the figure), pH=6.8 PBS (corresponding to CLG-CS-FAP@NIN in pH6.8PBS in the figure), pH=7.4 PBS (corresponding to CLG-CS-FAP@NIN in pH7.4 PBS in the figure)).

[0081] Depend on Figure 5 As can be seen in (a), the CLG-CS-FAP@NIN nanoformulation has no obvious change in particle size at 4°C, 25°C and 37°C, indicating that the CLG-CS-FAP@NIN nanoformulation has good stability under different conditions. Figure 5 As can be seen in (b), in the lung fluid or release medium with pH = 6.8, the drug release of the CLG-CS-FAP@NIN nanoformulation reached 70% at 48h, which was significantly higher than that of other groups, indicating that the CLG-CS-FAP@NIN nanoformulation prepared in the present invention can achieve efficient and sustained drug release in the weakly acidic microenvironment of the lungs, has obvious pH responsiveness, and helps to improve the utilization of the drug.

[0082] Effect Example 4 This effect example performs in vitro cytotoxicity tests on the CS@NIN, CLG-CS@NIN, CS-FAP@NIN, and CLG-CS-FAP@NIN nanoformulations prepared in Examples 1-4: The MTT method was used to detect the cytotoxicity of the above nanoformulations on various lung cells (BEAS-2B, RAW264.7, NIH-3T3, and NIH-3T3 induced by TGF-β), which specifically included the following steps: the logarithmic phase cells were plated on a 96-well plate at a density of 5000 / well, and the cells were cultured in a DMEM medium containing 10% serum. After 24 hours, the nanoformulations were prepared into different concentrations using a serum-free medium and added to a 96-well plate. After incubation for 24 hours, 20 μL of tetramethylthiazolyl blue solution (MTT, 5 mg / mL) was added to each well, and the plate was placed in an incubator for another 4 hours. The medium was discarded, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the OD value of each well at 490 nm was detected using an enzyme marker, and the cell survival rate was calculated based on the OD value.

[0083] The results are as follows Figure 6 As shown, for normal lung cells (BEAS-2B, RAW264.7), the nanoformulation prepared by the present invention has good in vitro safety; while for NIH-3T3 and NIH-3T3 induced by TGF-β, the cell survival rate decreased in a dose-dependent manner, and the cell activity decreased significantly with the increase of nintedanib concentration, indicating that the prepared nanoformulation has a good ability to inhibit the proliferation of fibroblasts and myofibroblasts in vitro.

[0084] Effect Example 5 This effect example tests the in vitro cellular uptake of the CS@NIN, CLG-CS@NIN, CS-FAP@NIN, and CLG-CS-FAP@NIN nanoformulations prepared in Examples 1-4: The uptake of the nanoformulation prepared by the present invention in in vitro cells (NIH-3T3, NIH-3T3 induced by TGF-β) was detected by confocal laser scanning microscopy (CLSM). In step S1 of Example 1, after each raw material component was dissolved in chloroform, a rhodamine solution (1 mg / mL) was added to the chloroform, and the mixture was protected from light. The remaining steps and parameters were the same as those of Examples 1-4, and a rhodamine (Rho)-labeled nanoformulation was prepared.

[0085] NIH-3T3 cells growing in logarithmic phase were taken and 1×10 5The cells / wells were plated in a confocal dish and placed in an incubator overnight. After the cells adhered to the wall, rhodamine (Rho)-labeled nanoformulations Rho-CS@NIN, Rho-CLG-CS@NIN, Rho-CS-FAP@NIN, and Rho-CLG-CS-FAP@NIN were added and incubated in an incubator in the dark for 4 hours. After 4 hours, the culture medium was discarded, the dish was washed twice with PBS, and then 4% paraformaldehyde was added to fix it for 15 minutes, and then washed twice with PBS, and DAPI was added for cell nucleus staining for 10 minutes. After washing twice with PBS, the dish was photographed and the cell uptake was observed under a laser confocal microscope.

[0086] NIH-3T3 cells growing in logarithmic phase were taken and 1×10 5 The cells / well were plated in a confocal dish, and 10 ng / mL of TGF-β was added to induce the transformation of fibroblasts into myofibroblasts. After culturing in an incubator for 48 h, rhodamine (Rho)-labeled nanoformulations Rho-CS@NIN, Rho-CLG-CS@NIN, Rho-CS-FAP@NIN, and Rho-CLG-CS-FAP@NIN were added and incubated in an incubator in the dark for 4 h. After 4 h, the culture medium was discarded, and the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, and DAPI was added for nucleus staining for 10 min. After washing twice with PBS, the cells were photographed and the cell uptake was observed under a laser confocal microscope.

[0087] The results are as follows Figure 7 As shown in (a) and (b), after 4 hours of incubation, uptake was visible in NIH-3T3 cells, but the fluorescence intensity was weak and basically the same in each group; and after TGF-β induction of NIH-3T3 cells, the fluorescence intensity of the Rho-CLG-CS@NIN group was significantly greater than that of the Rho-CS@NIN group, and the fluorescence intensity of the Rho-CS-FAP@NIN group was also greater than that of the Rho-CS@NIN group. The fluorescence intensity of the Rho-CLG-CS-FAP@NIN group was significantly stronger than that of the other three groups, with significant fluorescence intensity.

[0088] Therefore, the above experimental results show that the nanoformulation loaded with collagenase prepared in Example 2 can degrade collagen, break through the extracellular matrix barrier, and promote the cell uptake of the nanoformulation; and show that the nanoformulation loaded with FAP targeting peptide prepared in Examples 3 and 4 can specifically target myofibroblasts, bring the drug targeting to activated fibroblasts, and enable the drug to accurately exert its anti-fibrosis effect.

[0089] Effect Example 6 This effect example verifies the efficacy of the CS@NIN, CLG-CS@NIN, CS-FAP@NIN, and CLG-CS-FAP@NIN nanoformulations prepared in Examples 1-4 in a mouse pulmonary fibrosis model through animal experiments: 6.1 Establishment of mouse pulmonary fibrosis model First, a bleomycin-induced C57BL / 6 mouse pulmonary fibrosis model was established. The model was established by intratracheal instillation. The bleomycin concentration was 1.5 mg / kg. The mice were randomly divided into 7 groups, with 6 mice in each group, namely the normal group (Control), bleomycin group (BLM), nintedanib small molecule oral drug group (Free NIN), CS@NIN group, CLG-CS@NIN group, CS-FAP@NIN group, and CLG-CS-FAP@NIN group. Since the bleomycin-induced mouse pulmonary fibrosis model is in the transition period from pneumonia to pulmonary fibrosis in the first 1-7 days, and enters the pulmonary fibrosis stage after 7 days; therefore, this effect example chose to start aerosol administration treatment on the 8th day after modeling, which can more objectively reflect the therapeutic effect of the nanoformulation prepared by the present invention on the animal model of pulmonary fibrosis. The lung tissues of the untreated mice after modeling were taken for HE, Masson, and Sirius red staining. The results are as follows Figure 8 As shown in the figure, the mouse lung tissue showed obvious histopathological characteristics of fibrosis after modeling, including excessive deposition of interstitial collagen and destruction of alveolar structure. In the healthy lungs of the control group, the alveolar structure was intact, without collagen deposition or inflammatory infiltration, indicating that the mouse pulmonary fibrosis model was successfully established. After the modeling was successful, the drug was administered by aerosol every 2 days for a total of 7 times, and the dosage of nintedanib was 2.5 mg / kg each time.

[0090] 6.2 Plotting of mouse survival curves and monitoring of body weight changes Bleomycin was instilled into the trachea of ​​mice from day 0, and the weight of mice was measured after one day. The survival and weight changes of mice in each group after aerosol inhalation were compared. The results are as follows: Fig. 9 As shown, the weight of mice in the normal group without modeling was relatively stable, and the survival rate was 100%. The weight of mice that were not treated after modeling dropped sharply, and the survival rate dropped significantly to only about 25%. Compared with other drug-treated groups, the weight loss of mice in the CLG-CS-FAP@NIN group was significantly slower, and the survival rate was significantly improved to 85%, which was significantly better than the bleomycin group. This can explain to a certain extent that the CLG-CS-FAP@NIN nanoformulation prepared by the present invention has a good therapeutic effect on pulmonary fibrosis.

[0091] 6.3 Photography of mouse lung tissue and detection of hydroxyproline content On the 21st day after bleomycin modeling and aerosol administration, the mice were killed and the lung tissues were removed. The pathological morphology of the lung tissues was observed with the naked eye and photographed. The results are as follows: Fig.10As shown in the figure (the small picture below each group in the figure corresponds to the 5 lobes of the whole lung in the figure above), the lung tissue of the normal group of mice is ruddy in appearance, smooth in surface, and soft in texture; while the lung tissue of the bleomycin group of mice is accompanied by some bleeding spots, darkening in color, and poor elasticity; the lung tissue of the mice in the other drug-treated groups has improved to varying degrees, among which the bleeding spots of the lung tissue of the CLG-CS-FAP@NIN group of mice are significantly reduced, the color is basically close to that of the lung tissue of the normal group of mice, and the elasticity is basically restored. After the photo was taken, the ELISA assay kit was used to detect the content of hydroxyproline in the lung tissue homogenate of each group of mice. The results are shown in the figure below. Fig.11 As shown in (a), the hydroxyproline content in the lung tissue of mice in the bleomycin group was the highest, and the hydroxyproline content in other drug-administered groups decreased to a certain extent, among which the hydroxyproline content in the CLG-CS-FAP@NIN group was significantly less than that in other drug-administered groups. The above results indicate that the CLG-CS-FAP@NIN nanoformulation prepared by the present invention has a good therapeutic effect on pulmonary fibrosis after aerosol inhalation treatment.

[0092] 6.4 Counting the total number of cells in mouse bronchoalveolar lavage fluid On the 21st day after bleomycin modeling and aerosol administration, the mice were killed and the bronchoalveolar lavage fluid (BALF) of each group of mice was extracted. The supernatant was separated from the cell pellet after centrifugation. The cells were resuspended in PBS and then counted to detect the changes in the total number of cells in the alveolar lavage fluid. The results are as follows: Fig.11 As shown in (b), the total number of cells in the alveolar lavage fluid of mice after aerosol inhalation treatment is less than that in the alveolar lavage fluid of mice in the bleomycin group, and the total number of cells in the alveolar lavage fluid of mice in the CLG-CS-FAP@NIN group is significantly less than that in other administration groups, indicating that the CLG-CS-FAP@NIN nanoformulation in the present invention has a good therapeutic effect on pulmonary fibrosis after aerosol inhalation treatment. The separated supernatant was stored in a refrigerator at -80°C for subsequent ELISA inflammatory factor detection and BCA protein content detection.

[0093] 6.5 Biosafety Analysis of Nanoparticles in Mice On the 21st day after the nebulized administration of bleomycin to healthy mice according to the treatment regimen of model mice, the mice were killed, and the important organs such as the heart, liver, spleen and kidney of the mice were taken for HE staining to analyze the biosafety of the nanoformulation prepared by the present invention in vivo. The results are as follows: Fig.12 As shown, consistent with normal healthy mice, no obvious pathological changes were found in the tissues of mice after aerosol inhalation of the nanoformulation. The above results indicate that the nanoformulation prepared by the present invention has good biosafety.

[0094] In summary, the CLG-CS-FAP@NIN nanoformulation prepared in the present invention has good stability, slow drug release, good biosafety, the ability to break through the mucus and collagen barriers to reach the site of injury, and the ability to specifically target myofibroblasts. It has the ability to achieve active targeted and efficient treatment of pulmonary fibrosis with synergistic drugs after atomized inhalation.

[0095] The present invention constructs a composite drug-loaded nanoformulation for pulmonary administration by aerosol inhalation, co-modifies the surface of nintedanib-loaded nanoliposomes with FAP targeting peptide and collagenase, and prepares a composite nanoformulation (CLG-CS-FAP@NIN) with myofibroblast targeting and degradation of excess collagen in the extracellular matrix for the treatment of pulmonary fibrosis.

[0096] The present invention combines nanotechnology, targeted drug delivery and atomization inhalation technology, giving full play to the advantages of nanomedicine in the medical field. This fusion of multiple technologies brings new possibilities for the treatment of pulmonary fibrosis and improves the accuracy and efficacy of treatment.

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite drug-loaded nanoformulation, characterized in that: It comprises a drug-loaded nanoliposome, and a biological enzyme and a myofibroblast targeting ligand loaded on the surface of the drug-loaded nanoliposome; The drug-loaded nanoliposomes are nanoliposomes encapsulating anti-fibrosis drugs; The mass ratio of the drug-loaded nanoliposome to the biological enzyme and myofibroblast targeting ligand is (6-15): (0.5-4): (0-3).

2. The composite drug-loaded nanoformulation according to claim 1, characterized in that: The drug-loaded nanoliposomes include phospholipids, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide and anti-fibrosis drugs; the mass ratio of the phospholipids, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide and anti-fibrosis drugs is (10-25): (0-3): (0.5-5): (0.5-4); The phospholipid is at least one of egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, and distearoylphosphatidylethanolamine-polyethylene glycol.

3. The composite drug-loaded nanoformulation according to claim 2, characterized in that: The drug-loaded nanoliposome is prepared by a thin film hydration method, an ethanol injection method, an ammonium sulfate gradient method, a reverse evaporation method, a freeze drying method or an ultrasonic dispersion method.

4. The composite drug-loaded nanoformulation according to claim 1, characterized in that: The anti-fibrosis drug is at least one of nintedanib, pirfenidone, metformin, resveratrol, curcumin, and trametinib.

5. The composite drug-loaded nanoformulation according to claim 1, characterized in that: The biological enzyme is collagenase.

6. The composite drug-loaded nanoformulation according to claim 1, characterized in that: The myofibroblast targeting ligand is a FAP targeting peptide.

7. A method for preparing the composite drug-loaded nanoformulation according to any one of claims 1 to 6, characterized in that: The following steps are involved: The drug-loaded nanoliposomes and the myofibroblast targeting ligand were reacted under nitrogen sealing and dialyzed to obtain nanoformulation A; Adding the nano preparation A to the chitosan solution for reaction and dialyzing to obtain the nano preparation B; The nano preparation B is reacted with a biological enzyme and dialyzed to obtain the composite drug-loaded nano preparation.

8. The method for preparing the composite drug-loaded nanoformulation according to claim 7, characterized in that: The concentration of chitosan in the chitosan solution is 0.1-2 mg / mL, and the solvent of the chitosan solution is an acetic acid solution with a volume concentration of 0.5-5%.

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