Preparation method and application of inhalation drug-loading ultrasonic driving nano motor

Abstract

The invention aims to provide a preparation method and application of an inhalation drug-loading ultrasonic driving nano motor, and can effectively solve the problems of preparation of the inhalation drug-loading ultrasonic driving nano motor and application of the inhalation drug-loading ultrasonic driving nano motor in preparation of drugs for treating IPF. Comprising the following steps: firstly preparing PLGA-PFD nanoparticles, then preparing PDA-PLGA-PFD nanoparticles, and finally preparing BNN6 / PDA-PLGA-PFD, namely the asymmetric nano motor BPPP with the ultrasonic responsiveness. The product disclosed by the invention is scientific and advanced in preparation method, good in product performance, high in atomization administration and IPF lung pathological barrier penetrating capability, high in medicine utilization rate, good in curative effect, simple and convenient to operate, wide in application prospect and huge in economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a preparation method and application of an inhalation drug-loaded ultrasound-driven nanomotor. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease. Currently, high-dose pirfenidone oral treatment is mainly used clinically, but there are side effects such as hepatotoxicity and phototoxicity. Inhalation administration can reduce the dosage and improve the targeting. However, the inhaled drug delivery system is limited by the thickened mucus layer and the deposited collagen barrier. The existence of the pathological barrier makes it difficult for the drug to reach the fibroblast target. The development of a nanosystem with mucus penetration function through the chemical composition and charge characteristics of mucin provides technical support for the drug to cross the mucus barrier. However, the overall penetration efficiency obtained by passive diffusion of nanoparticles is still limited. Nanomotors can utilize the energy in the environment to achieve autonomous propulsion. Among them, the ultrasound-driven nanomotor combines non-invasive characteristics and excellent active diffusion performance, showing significant advantages in drug delivery. However, after crossing the pulmonary mucus barrier, the inhaled drug delivery system will still face a dense and hard deposited collagen barrier, which limits the drug delivery in the lung interstitial tissue. The existing nanomotors have a single function and are difficult to cross complex physiological barriers such as the dense deposited collagen in the IPF lesion site.

[0003] Nitric oxide (NO) is a bioactive gas that can rapidly react with endogenous reactive oxygen species at the inflammatory site to generate peroxynitrite ion (ONOO - ), ONOO -It can rapidly activate precursor matrix metalloproteinases (Pro-MMPs) in the extracellular matrix (ECM) to generate active matrix metalloproteinases (MMPs), thereby efficiently promoting the degradation of collagen deposited in the ECM. Using clinically approved poly(lactic acid-co-glycolic acid) (PLGA) as a carrier, a drug-loaded unit (PP) of pirfenidone (PFD) was prepared. Hydrophilic polyacrylic acid (PAA) was adsorbed onto hydrophobic PP nanoparticles. Subsequently added dopamine would preferentially polymerize at the PAA site and achieve anisotropic growth, in-situ growing into asymmetric nanoparticles PPP. Finally, the ultrasound-responsive NO donor N,N'-di-sec-butyl-N,N'-dinitro-p-phenylenediamine (BNN6) was selectively loaded on one side of the polydopamine hemisphere (PDA) through π-π conjugation to prepare an ultrasound self-propelled nanomotor (BPPP). After BPPP was inhaled by atomization, under highly efficient and controllable ultrasound stimulation, NO gas could be generated unidirectionally on the surface of the nanomotor, thereby forming a bubble driving force to promote the efficient crossing of the nanosystem across the pulmonary mucus barrier. At the same time, NO rapidly reacted with reactive oxygen species (ROS) in the inflammatory pathological microenvironment to generate ONOO⁻, and ONOO⁻ activated Pro-MMPs in the extracellular matrix into active MMPs. MMPs further degraded the collagen deposited at the fibrotic site, clearing the way for the nanosystem to reach the diseased fibroblasts and improving the drug delivery efficiency. However, there has been no public report on how to prepare an inhaled drug-loaded ultrasound-driven nanomotor and use it to prepare a drug for the treatment of IPF. Summary of the Invention

[0004] In view of the above situation, to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of an inhaled drug-loaded ultrasound-driven nanomotor, which can effectively solve the preparation of the inhaled drug-loaded ultrasound-driven nanomotor and realize its application in the preparation of drugs for the treatment of IPF.

[0005] The technical solution adopted by the present invention to solve the problem is that a preparation method of an inhaled drug-loaded ultrasound-driven nanomotor includes the following steps: 1) Preparation of PLGA-PFD (PP) nanoparticles: Poly(lactic acid-co-glycolic acid) (PLGA) and pirfenidone (PFD) were dissolved in dichloromethane at a mass ratio of 4:1 to form a mixed solution. Then the mixed solution was added to a polyvinyl alcohol solution (PVA), and a water-in-oil (O / W) emulsion was formed by a probe sonicator. The emulsion was added to deionized water, stirred overnight, the remaining dichloromethane was evaporated, centrifuged, and washed with water to obtain PLGA-PFD (PP) nanoparticles; At the carboxyl end of the poly(lactic-co-glycolic acid) (PLGA) described, PLGA is prepared by ring-opening polymerization. Glycolic acid and lactic acid are dehydrated and cyclized respectively to synthesize two monomers, lactide (LA) and glycolide (GA). Then, LA and GA are ring-opening polymerized in a mass ratio of 65:35 to obtain poly(lactic-co-glycolic acid) (PLGA) (a well-known technology).

[0006] 2) Preparation of PDA-PLGA-PFD (PPP) nanoparticles: First, disperse the PP nanoparticles in deionized water, add ammonia water, then dropwise add polyacrylic acid (PAA) and stir to serve as the "seed" for initiating the polymerization of polydopamine (PDA). Then, dropwise add isopropanol, and then dropwise add ammonia water again. Next, drop the dopamine aqueous solution into the mixed system, react, and centrifuge to collect the nanoparticles, which are washed twice to prepare PDA-PLGA-PFD (PPP) nanoparticles with an asymmetric structure; 3) Preparation of BNN6 / PDA-PLGA-PFD (nanomotor BPPP): A. Add the N,N-di-sec-butyl-p-phenylenediamine solution to ethanol. Under a nitrogen atmosphere, slowly add the sodium nitrite solution. After the reaction, slowly drop hydrochloric acid into the reaction system, and stir the reaction under nitrogen protection and in the dark. A pale yellow precipitate is formed, centrifuged, washed to remove the residues, and freeze-dried to obtain the ultrasound-sensitive nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-p-phenylenediamine (BNN6), which is stored in the dark; B. Disperse BNN6 and PPP nanoparticles in acetonitrile and stir in the dark. BNN6 is adsorbed on the surface of the PPP nanoparticles through π-π conjugation. Centrifuge to obtain a precipitate, which is washed with water to obtain the ultrasound-responsive asymmetric nanomotor BPPP.

[0007] Application of the inhaled drug-loaded ultrasound-driven nanomotor BPPP prepared by the method of the present invention in the preparation of drugs for treating IPF.

[0008] The preparation method of the product of the present invention is scientific and advanced, the product has good performance, is atomized for drug delivery, has strong ability to penetrate the pulmonary pathological barrier of IPF, high drug utilization rate, good curative effect, is simple and convenient to operate, has broad application prospects, and huge economic and social benefits. Description of the Drawings

[0009] Figure 1 It is the TEM result diagram of the PP of the present invention.

[0010] Figure 2 It is the TEM result diagram of the PPP of the present invention.

[0011] Figure 3 It is the elemental mapping result diagram of the PPP of the present invention.

[0012] Figure 4 Curves of NO release by BPPP of the present invention under different power ultrasounds.

[0013] Figure 5 Curves of MSD results of BPPP of the present invention.

[0014] Figure 6 Result graphs of the effects of the nanocarriers of the present invention on the viability of A549 cells respectively.

[0015] Figure 7 Result graphs of the effects of the nanocarriers of the present invention on the viability of WML-2 cells respectively. Detailed implementation manners

[0016] The following combines examples and specific situations to detail the specific implementation manners of the present invention. Examples

[0017] The technical solution of the present invention is a preparation method of an inhaled drug-loaded ultrasound-driven nanomotor, including the following steps: 1) Preparation of PLGA-PFD (PP) nanoparticles: Dissolve 20 mg of poly (lactic-co-glycolic acid) (PLGA) and 5 mg of pirfenidone (PFD) (i.e., the mass ratio of PLGA to PFD is 4:1) in 400 μL of dichloromethane to form a mixed solution, and then add the mixed solution to 3 ml of a 5% (volume concentration) polyvinyl alcohol solution (Polyvinyl alcohol, PVA). Use a probe sonicator to sonicate on ice for 1 min, with 4 s on and 2 s off in the middle to form an oil-in-water (O / W) emulsion; add the emulsion to 5 mL of deionized water, stir overnight for 8 - 12 h, evaporate the remaining dichloromethane, centrifuge at 10000 rpm for 30 min at 4°C, and then wash 2 times by centrifugation at 10000 rpm and redispersion with water to obtain PLGA-PFD (PP) nanoparticles; For the carboxyl end of the poly (lactic-co-glycolic acid) (PLGA), PLGA is prepared by ring-opening polymerization. Glycolic acid and lactic acid are dehydrated and cyclized respectively to synthesize two monomers, lactide (LA) and glycolide (GA), and then LA and GA are ring-opening polymerized in a mass ratio of 65:35 to obtain poly (lactic-co-glycolic acid) (PLGA).

[0018] 2) Preparation of PDA-PLGA-PFD (PPP) nanoparticles: First, disperse 10 mg of PP nanoparticles in 10 mL of deionized water, add 150 μL of 2 M ammonia water, then dropwise add 100 μL of polyacrylic acid (PAA) and stir for 10 min to serve as the "seed" for initiating the polymerization of polydopamine (PDA). Then, dropwise add 40 mL of isopropanol at a dropping rate of 100 μL / min. Subsequently, dropwise add 500 μL of ammonia water at a dropping rate of 100 μL / min. Then, drop 200 - 400 μL of dopamine aqueous solution with a concentration of 50 mg / mL into the mixed system and react at 50 °C for 3 h. Centrifuge at 12,000 rpm to collect the nanoparticles, and wash them alternately with 20 mL of ultrapure water and 20 mL of absolute ethanol twice, 15 min each time, to prepare PDA-PLGA-PFD (PPP) nanoparticles with an asymmetric structure; 3) Preparation of BNN6 / PDA-PLGA-PFD (nano-motor BPPP): A. Accurately measure 2.34 mL of N,N-di-sec-butyl-p-phenylenediamine solution with a concentration of 0.4149 g / mL, add it to 20 mL of ethanol. Under a nitrogen atmosphere, slowly add 20 mL of 6 M sodium nitrite solution to the reaction system. After reacting for 30 min, slowly drop 20 mL of 6 M hydrochloric acid into the reaction system, and stir and react for 4 h under nitrogen protection and light avoidance conditions. The reaction system changes from red to orange, and finally a pale yellow precipitate is formed. Centrifuge at 6,500 rpm for 15 min at 4 °C, then wash it alternately with 20 mL of ultrapure water and 20 mL of 50% (v / v) ethanol aqueous solution three times, 15 min each time, to remove the residues, and freeze-dry to obtain the ultrasound-sensitive nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-p-phenylenediamine (BNN6), and store it in the dark at -20 °C; B. Disperse BNN6 and PPP nanoparticles in 10 mL of 10% (v / v) acetonitrile. The mass ratio of BNN6 to PPP nanoparticles is 1:5, and stir for 24 h in the dark. BNN6 is adsorbed on the surface of PPP nanoparticles through π-π conjugation. Then centrifuge at 5,000 rpm for 10 min to obtain a precipitate, and wash the precipitate three times with water to obtain the ultrasound-responsive asymmetric nano-motor BPPP. Example

[0019] In the preparation method of the oral drug-loaded dual-driven nano-motor of the present invention, the preferred addition volume of the dopamine aqueous solution in the preparation process of the PPP nanoparticles is 250 μL, and the particle size of the inhaled drug-loaded ultrasound-driven nano-motor is 100 - 120 nm; Application of the inhaled drug-loaded ultrasound-driven nano-motor BPPP prepared by the method of the present invention in the preparation of drugs for treating IPF.

[0020] According to the preparation method given in the above embodiments, any amount of nano-motor BPPP can be prepared as needed. The given embodiments are only used to illustrate the specific implementation manners of the present invention, rather than to limit the protection scope of the present invention. Any technical solutions that are essentially the same as the present application made by means of equivalent substitution or transformation means all fall within the protection scope of the present invention.

[0021] The preparation method of the product of the present invention is scientific and advanced, the product has good performance, is atomized for drug delivery, has strong ability to penetrate the pathological barrier of the IPF lung, high drug utilization rate, good curative effect, safe medication, and has obtained very good beneficial technical effects through experiments. The relevant data are as follows: Physical characteristic expression

[0022] The present invention uses a transmission electron microscope to analyze the morphology of PLGA-PFD, with a scale of 100 nm. The results are as Figure 1 shown. It can be analyzed from Figure 1 that the average size of PLGA nanoparticles is about 75 nm; Use a transmission electron microscope to analyze the morphology of PPP asymmetric nanoparticles, with a scale of 100 nm. The results are as Figure 2 , 3 shown; It can be analyzed from Figure 2 , 3 that the average particle size of PPP nanoparticles is 110 nm; Use a particle size analyzer to perform hydrodynamic analysis on the potential level and particle size distribution of the inhaled drug-loaded ultrasonic-driven nano-motor BPPP, and use a transmission electron microscope to analyze its morphology. As Figure 4 shown, the potential of PP is -21.1 mV, the potential of PPP is -35.9 mV, the surface charge of BPPP after loading BNN6 is -34.8 mV, and the particle size is 100 - 120 nm; In vitro characteristic experiments of BPPP

[0023] 1) NO release efficiency of BPPP nano-motor Specific experimental method: Disperse 20.4 μg / mL of BNN6 and 197.8 μg / mL of BPPP in phosphate buffer (PBS) respectively, and irradiate them for 10 minutes under an external ultrasound of 0, 0.2, 0.4, and 0.6 W / cm² respectively. Subsequently, collect 50 μL of the supernatant obtained by centrifugation for quantitative detection of the released NO. Then place the suspension in the dark for 20 minutes, and take out 50 μL of the supernatant every 10 minutes for measurement. After reacting with Greiss reagent, immediately measure the absorption of the collected supernatant at a wavelength of 540 nm by an enzyme-linked immunosorbent assay to detect the release rate of NO.

[0024] The results are as Figure 5As shown, NO can only be released during ultrasonic stimulation, and when the ultrasonic power is 0.6 W / cm², the NO release amount of BPPP is 15.8 μM. -2

[0025] 2) Investigation experiment on the autonomous motility of BPPP nanomotors Under a confocal microscope, the movement of particles in mucus was analyzed using the multi-particle tracking (MPT) method. 197.8 μg of BPPP was added to 1 mL of mucin solution and mixed evenly. The movement trajectories within 10 s under external ultrasounds of 0, 0.2, 0.4, and 0.6 W / cm² were investigated respectively, and the mean squared displacement (MSD) of the nanoparticles at different powers was analyzed.

[0026] The results are as Figure 6 shown. With the increase of ultrasonic power, the MSD of BPPP increased significantly, indicating that this inhalation drug-loaded ultrasound-driven nanomotor has good autonomous motility.

[0027] 3) Investigation experiment on the in vitro biosafety of BPPP nanomotors A549 cells and WML-2 cells in good growth state were seeded in 96-well plates at a density of 8×10 3 / well, with 3 replicates in each group, and then incubated overnight for 8 - 12 h in an incubator; washed 3 times with PBS for 2 min each time, and different concentrations (50, 10, 200, 400, 600 μg / mL) of PPP + US, BNN6, BPPP, and BPPP + US were added to the well plates respectively, and incubated for another 24 h. After washing 3 times with PBS for 2 min each time, 10 μL of CCK8 and 90 μL of medium were added to each well, mixed evenly, and after continuing to incubate for 3 h, the absorbance at OD450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated to evaluate the in vitro safety.

[0028] The results are as Figure 7 shown. When the nanocarriers were incubated with the two types of cells at different concentrations for 24 h, the cell viability was above 90%. After external ultrasound (0.6 W / cm²; 5 min), there was no obvious toxicity either. It was proved that this nanocarrier has good biosafety. -2

[0029] In summary, the present invention provides a method for preparing an inhalation drug-loaded ultrasound-driven nanomotor and its application in the preparation of drugs for idiopathic pulmonary fibrosis to solve the problem that drugs are difficult to cross the pathological lung barrier. Using clinically approved PLGA as a carrier, a drug-loaded unit (PP) of PFD is prepared. Hydrophilic PAA is adsorbed on the hydrophobic PP nanoparticles. Subsequently added dopamine will preferentially polymerize at the PAA site and achieve anisotropic growth, in-situ growing into asymmetric nanoparticles PPP. Finally, the ultrasound-responsive nitric oxide donor BNN6 is selectively loaded on one side of PDA to prepare the bioactive self-driven nanomotor BPPP. After BPPP is nebulized and inhaled, under highly efficient and controllable ultrasound stimulation, NO gas can be generated unilaterally on the surface of the nanomotor, thereby forming a bubble driving force to promote the efficient crossing of the lung mucus barrier by the nanosystem. At the same time, NO quickly reacts with ROS in the inflammatory pathological microenvironment to generate ONOO⁻, and ONOO⁻ activates Pro MMPs in the extracellular matrix into active MMPs. MMPs further degrade the collagen deposited at the fibrotic site, clearing the way for the nanosystem to reach the diseased fibroblasts, and improving the drug delivery efficiency and efficacy.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) The inhalation drug-loaded ultrasound-driven nanomotor of the present invention has rich sources of synthetic materials, good safety, low cost, is convenient for clinical transformation, and has practical clinical application value; 2) The inhalation drug-loaded ultrasound-driven nanomotor provided by the present invention realizes the active penetration through the thickened mucus layer in the alveolar area of idiopathic pulmonary fibrosis after nebulized inhalation by means of the bubble driving force of NO.

[0031] 3) The inhalation drug-loaded ultrasound-driven nanomotor provided by the present invention solves the limitation of the inhaled nano-drug delivery system by the dense deposited collagen at the idiopathic pulmonary fibrosis lesion site. The whole system has the advantages of safe medication, good water solubility, strong stability, good biocompatibility, and no obvious toxic and side effects; using the ultrasound-driven nanomotor, it realizes the efficient penetration of the mucus and collagen barriers, and finally realizes the delivery of drugs to lung fibroblasts, with significant anti-fibrotic ability. It is a major innovation in drugs for treating idiopathic pulmonary fibrosis, has the value of popularizing practical applications, and has great economic and social benefits.

Claims

1. A preparation method of an inhaled drug-loaded ultrasound-driven nanomotor, comprising the following steps: 1) Preparation of PLGA-PFD (PP) nanoparticles: Dissolve 20 mg of poly (lactic-co-glycolic acid) (PLGA) and 5 mg of pirfenidone (PFD) (i.e., the mass ratio of PLGA to PFD is 4:1) in 400 μL of dichloromethane to form a mixed solution. Then add the mixed solution to 3 mL of a 5% (v / v) polyvinyl alcohol (PVA) solution, and ultrasonicate with a probe sonicator on ice for 1 min, with 4 s on and 2 s off in the middle to form an oil-in-water (O / W) emulsion. Add the emulsion to 5 mL of deionized water, stir overnight for 8 - 12 h, evaporate the remaining dichloromethane, centrifuge at 10000 rpm for 30 min at 4°C, and then wash twice with water by 10000 rpm centrifugation and redispersion cycles to obtain PLGA-PFD (PP) nanoparticles; For the carboxyl end of the poly (lactic-co-glycolic acid) (PLGA), PLGA is prepared by ring-opening polymerization. Glycolic acid and lactic acid are dehydrated and cyclized respectively to synthesize two monomers, lactide (LA) and glycolide (GA). Then LA and GA are ring-opening polymerized in a mass ratio of 65:35 to obtain poly (lactic-co-glycolic acid) (PLGA); 2) Preparation of PDA-PLGA-PFD (PPP) nanoparticles: First, disperse 10 mg of PP nanoparticles in 10 mL of deionized water, add 150 μL of 2M ammonia water, and then dropwise add 100 μL of polyacrylic acid (PAA) and stir for 10 min as the "seed" for initiating the polymerization of polydopamine (PDA). Then dropwise add 40 mL of isopropanol at a dropping rate of 100 μL / min, and then dropwise add 500 μL of ammonia water at a dropping rate of 100 μL / min. Then drop 200 - 400 μL of a 50 mg / mL dopamine aqueous solution into the mixed system and react at 50°C for 3 h. Centrifuge at 12000 rpm to collect the nanoparticles, and wash twice alternately with 20 mL of ultrapure water and 20 mL of absolute ethanol to prepare PDA-PLGA-PFD (PPP) nanoparticles with an asymmetric structure; 3) Preparation of BNN6 / PDA-PLGA-PFD (nanomotor BPPP): A. Accurately measure 2.34 mL of N,N-di-sec-butyl-p-phenylenediamine solution with a concentration of 0.4149 g / mL, add it to 20 mL of ethanol. Under a nitrogen atmosphere, slowly add 20 mL of 6 M sodium nitrite solution to the reaction system. After reacting for 30 min, slowly drip 20 mL of 6 M hydrochloric acid into the reaction system, and stir and react for 4 h under nitrogen protection and in the dark. The reaction system changes from red to orange, and finally a pale yellow precipitate is formed. Under the condition of 4 °C, centrifuge at 6500 rpm for 15 min, then wash it alternately with 20 mL of ultrapure water and 20 mL of 50% ethanol aqueous solution by volume for 3 times, 15 min each time, to remove the residues, and freeze-dry to obtain the ultrasound-sensitive nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-p-phenylenediamine (BNN6), and store it in the dark at -20 °C; B. Disperse BNN6 and PPP nanoparticles in 10 mL of 10% acetonitrile by volume. The mass ratio of BNN6 to PPP nanoparticles is 1:5, and stir for 24 h in the dark. BNN6 is adsorbed on the surface of PPP nanoparticles through π-π conjugation, and then centrifuge at 5000 rpm for 10 min to obtain a precipitate. Wash the precipitate with water three times to obtain the ultrasound-responsive asymmetric nanomotor BPPP.

2. The preparation method of the inhalation drug-loaded ultrasound-driven nanomotor according to claim 1, characterized in that In the preparation process of the PPP nanoparticles described above, the preferred volume of dopamine added is 250 μL, and the particle size of the inhaled drug-loaded ultrasound-driven nanomotor is 100 - 120 nm.

3. Use of the inhaled drug-loaded ultrasound-driven nanomotor prepared by the method according to claim 1 or 2 in the preparation of a drug for treating IPF.

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