Lung targeting nano-micelle, preparation method and application

Abstract

The invention provides a lung-targeted nano-micelle, a preparation method and application, and the lung-targeted nano-micelle comprises permanent cationic lipid, pH responsive lipid and phenylboronic acid modified cationic lipid. The molar ratio of the permanent cationic lipid to the pH-responsive lipid to the phenylboronic acid modified cationic lipid is X: 3-X: 1, and X is equal to 1-2, so that the nano-beam is regulated to target the lung.

Description

Lung-targeted nanomicelles, preparation methods, and applications Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and relates to a lung-targeted nanomicelle, a preparation method and an application thereof. Background Art

[0002] Lung disease is one of the most common diseases that threaten human life and health. Bacterial pneumonia is a common lung disease. Patient compliance with nebulized and oral administration is poor, resulting in suboptimal treatment outcomes. Intravenous antibiotics are a relatively effective treatment, but the emergence of multidrug-resistant bacteria and the challenges of antibiotic administration include poor targeting, low utilization, and short lung residence time. This makes lung targeting impossible, leading to poor treatment outcomes. Frequent, high-dose antibiotic use can lead to drug resistance and a range of toxic side effects, such as hepatotoxicity and renal toxicity and intestinal dysbiosis. Therefore, the development of lung-targeted nanomicelles is of great clinical significance in the treatment of lung diseases.

[0003] Summary of the Invention

[0004] In response to the above technical problems, the present disclosure provides a lung-targeted nanomicelle, a preparation method and an application thereof, in order to at least partially solve the above-mentioned technical problems.

[0005] In order to solve the above technical problems, the present disclosure provides the following technical solutions:

[0006] As a first aspect of the present disclosure, a lung-targeting nanomicelle is provided, comprising:

[0007] permanent cationic lipids;

[0008] pH-responsive lipids; and

[0009] Phenylboronic acid-modified cationic lipids;

[0010] Among them, the molar ratio of permanent cationic lipids, pH-responsive lipids, and phenylboronic acid-modified cationic lipids is X:3-X:1, where X=1~2, so that the nanomicelles are regulated to target the lungs.

[0011] In one embodiment, the permanent cationic lipid has the structure shown in Formula I:

[0012] Wherein, R1 is a C12-C18 alkyl group.

[0013] In one embodiment, the pH-responsive lipid has the structure shown in Formula II:

[0014] Wherein, R2 is a C12-C18 alkyl group.

[0015] In one embodiment, the phenylboronic acid-modified cationic lipid has the structure shown in Formula III:

[0016] Wherein, R3 is a C12-C18 alkyl group.

[0017] In one embodiment, the average particle size of the lung-targeted nanomicelles is 95.44±17.26 nm.

[0018] In one embodiment, the zeta potential of the lung-targeting nanomicelles is -21.49 to +20.91 mV.

[0019] As a second aspect of the present disclosure, a method for preparing the above-mentioned lung-targeting nanomicelles is provided, comprising:

[0020] The permanent cationic lipid, the pH-responsive lipid, and the cationic lipid modified with phenylboronic acid are mixed and dissolved in an organic solvent to obtain a mixed solution;

[0021] removing the organic solvent from the mixed solution to form a lipid film, and drying the lipid film to obtain a dried lipid film;

[0022] The dried lipid film was hydrated with ultrapure water to obtain a lipid suspension, which was then subjected to water bath ultrasonic treatment to form lung-targeted nanomicelles.

[0023] In one embodiment, the organic solvent includes at least one of methanol and chloroform.

[0024] In one embodiment, the method for removing the organic solvent is vacuum rotary evaporation, the speed of the vacuum rotary evaporation is 80 to 150 rpm, and the temperature of the vacuum rotary evaporation is 30 to 60° C.

[0025] The vacuum drying temperature is 20-30°C, and the vacuum drying time is 1.5-5h;

[0026] The frequency of ultrasonic treatment is 30-60 kHz, and the time of ultrasonic treatment is 1-10 minutes.

[0027] As a third aspect of the present disclosure, a pharmaceutical composition is provided, comprising the above-mentioned lung-targeting nanomicelles as a drug carrier.

[0028] As a fourth aspect of the present disclosure, there is provided a use of the above-mentioned lung-targeted nanomicelles in the preparation of a drug for treating lung diseases.

[0029] In one embodiment, the lung disease is pneumonia.

[0030] The lung-targeted nanomicelles, preparation methods, and applications disclosed herein are constructed from three lipids: a permanent cationic lipid, a pH-responsive lipid, and a phenylboronic acid-modified cationic lipid. By adjusting the ratio of the permanent cationic lipids, the surface charge properties of the nanomicelles can be regulated. As the ratio of the permanent cationic lipids changes, the surface charge of the nanomicelles changes from negative to positive, increasing the adsorption of targeted lung microenvironment proteins and achieving targeted delivery to the lungs. These nanomicelles can be used in the respiratory system, providing a potential opportunity for targeted drug delivery to lung tissue, reducing drug dosage, and effectively reducing the toxic side effects of drug treatments on other tissues, showing great clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 is a schematic diagram of the synthesis of permanent cationic lipids and pH-responsive lipids in Example 1 of the present disclosure;

[0032] FIG2 is a schematic diagram of the synthesis of a cationic lipid modified with phenylboronic acid in Example 1 of the present disclosure;

[0033] FIG3 is an electrospray ionization mass spectrum (ESI-MS) of the permanent cationic lipid in Example 1 of the present disclosure;

[0034] FIG4 is an electrospray ionization mass spectrometry (ESI-MS) diagram of the pH-responsive lipid in Example 1 of the present disclosure;

[0035] FIG5 is an electrospray ionization mass spectrometry (ESI-MS) diagram of the cationic lipid modified with phenylboronic acid in Example 1 of the present disclosure;

[0036] FIG6 is a diagram showing the particle size of lung-targeted nanomicelles in Example 2 of the present disclosure;

[0037] FIG7 is a Zeta potential diagram of the lung-targeting nanomicelles in Example 2 of the present disclosure;

[0038] FIG8 is a transmission electron microscope (TEM) image of the lung-targeting nanomicelles in Example 3 of the present disclosure;

[0039] FIG9 is a graph showing the particle size stability test of the lung-targeting nanomicelles in Example 3 of the present disclosure;

[0040] FIG10 is a graph showing the Zeta potential stability test of the lung-targeting nanomicelles in Example 3 of the present disclosure;

[0041] FIG11 is an imaging distribution diagram of the lung-targeting nanomicelles labeled with DiD fluorescence in Example 4 of the present disclosure;

[0042] FIG12 is a confocal laser scanning microscope (CLSM) image of the lung-targeting nanomicelles labeled with DiD fluorescence in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0044] Currently, amphiphilic liposomes are widely used as drug carriers. Their designability allows for targeted delivery and sustained release of loaded drugs, minimizing drug side effects and optimizing their pharmacokinetics. However, upon entering the bloodstream, traditional liposomes absorb large amounts of native immunoglobulin M (IgM), exacerbating immune system recognition and leading to accumulation in the liver and spleen. Consequently, most liposome-delivered drugs accumulate in the liver and spleen, preventing effective delivery to target organs.

[0045] In the process of realizing the present disclosure, it was discovered that the related technology can prepare a series of nucleic acid complexes with different surface charges by adjusting the ratio of phospholipids, cholesterol, polyethylene glycol-modified liposomes and cationic liposomes. Adjusting the ratio of cationic liposomes can achieve targeted delivery of nucleic acid complexes to different organs. As the ratio of cationic liposomes changes, the transported luciferase can be selectively expressed in the liver, spleen and lungs. However, the related technology for lung targeted delivery only appears in the field of gene therapy, and there are few reports on drugs for targeted delivery to the lungs. In view of the technical problems existing in the related technology, the present disclosure provides a lung-targeted nanomicelle composed of permanent cationic lipids, pH-responsive lipids and phenylboronic acid-modified cationic lipids. By adjusting the composition ratio of liposomes, especially the ratio of permanent cationic liposomes in the micelles, targeted delivery to the lungs can be achieved.

[0046] In order to achieve the above technical objectives, as a first aspect of the present disclosure, a lung-targeting nano-micelle is provided, comprising:

[0047] permanent cationic lipids;

[0048] pH-responsive lipids; and

[0049] Phenylboronic acid-modified cationic lipids;

[0050] Among them, the molar ratio of permanent cationic lipids, pH-responsive lipids, and phenylboronic acid-modified cationic lipids is X:3-X:1, where X=1~2, so that the nanomicelles are regulated to target the lungs.

[0051] According to the embodiments of the present disclosure, permanent cationic lipids are used to regulate the surface charge of nanomicelles, thereby regulating the adsorption performance of the charge on proteins in the blood; pH-responsive lipids are liposomes with pH-responsive function, which can achieve bacterial targeting through amide bonds in a slightly acidic bacterial environment, and can achieve charge reversal in a weakly acidic microenvironment, targeting cells with negative charges on the surface through electrostatic interactions; the diol structure in the cationic lipids modified with phenylboronic acid has bacterial targeting effect, which assists the pH-responsive lipid in unfolding. The molar ratio of the permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid constituting the nanomicelles can be 1:2:1, 1.2:1.8:1, 1.5:1.5:1, 1.8:1.2:1, 2:1:1, etc. The nanomicelles are regulated according to this ratio to target the lungs, and the ratio of the phenylboronic acid-modified cationic lipid remains unchanged. When the molar ratio of the permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 2:1:1, the surface charge of the obtained nanomicelles is neutral to positive, which is conducive to the adsorption of vitronectin in the serum. After reaching the lungs through the blood circulation, it specifically binds to the αvβ3 homologous receptor highly expressed in the lungs, thereby increasing the enrichment and retention time of the drug in the lungs, thereby completing lung targeting.

[0052] According to an embodiment of the present disclosure, the permanent cationic lipid has the structure shown in Formula I:

[0053] Among them, R1 is a C12-C18 alkyl group, which is used to regulate the charge, thereby regulating the adsorption of proteins in the blood by the nanomicelles, so that the nanomicelles are regulated to be lung-targeted.

[0054] According to an embodiment of the present disclosure, the method for preparing permanent cationic lipids is as follows:

[0055] N-tert-butyloxycarbonyl-1,2-ethylenediamine and C12-C18 alkyl bromide are reacted in the presence of a base catalyst to obtain Wherein, R1 is a C12-C18 alkyl group.

[0056] React with methyl halide to obtain the product Wherein, R1 is a C12-C18 alkyl group.

[0057] Then the tert-butyl carbonyl (BOC) of the above product is completely deprotected to obtain a permanent cationic lipid. Wherein, R1 is a C12-C18 alkyl group.

[0058] According to an embodiment of the present disclosure, the pH-responsive lipid has the structure shown in Formula II:

[0059] Among them, R2 is a C12-C18 alkyl group with a negative charge. The liposome has pH-responsive function and can achieve bacterial targeting through amide bonds in a slightly acidic bacterial environment.

[0060] According to an embodiment of the present disclosure, the method for preparing pH-responsive lipids is as follows:

[0061] The tert-butyl (2-aminoethyl) carbamate compound and C12-C18 alkyl bromide are reacted in the presence of a base catalyst to obtain Wherein, R2 is a C12-C18 alkyl group.

[0062] Take it off again The pH-responsive lipid was obtained by adding maleic anhydride to the tert-butyl carbonyl (BOC) Wherein, R2 is a C12-C18 alkyl group.

[0063] According to an embodiment of the present disclosure, the cationic lipid modified with phenylboronic acid has a structure shown in Formula III:

[0064] Where R3 is a C12-C18 alkyl group. The phenylboronic acid-modified cationic lipid has a neutral charge and utilizes the covalent bond between the boronic acid group and the cis-diol of the bacterial polysaccharide to achieve precise targeting of bacterially infected microdomains in the lungs. The reactive oxygen species-responsive phenylboronic acid-modified cationic lipid, together with the pH-responsive lipid, assists the pH-responsive lipid in unfolding its function. Furthermore, the microacidity and reactive oxygen species stimulation in the bacterially infected microdomains induce a structural transformation in the nanomicelles, rapidly releasing the loaded antibiotic.

[0065] According to an embodiment of the present disclosure, the method for preparing a phenylboronic acid-modified cationic lipid is as follows:

[0066] 2-aminoethanol and C12-C18 alkyl bromide react under the action of an alkali catalyst to obtain the product alkyl ethanol Wherein, R3 is a C12-C18 alkyl group.

[0067] Then Reaction with 4-(bromomethyl)phenylboronic acid to obtain phenylboronic acid-modified cationic lipids Wherein, R3 is a C12-C18 alkyl group.

[0068] According to an embodiment of the present disclosure, the average particle size of the lung-targeted nanomicelles is 95.44±17.26 nm, and the particle size of the lung-targeted nanomicelles can be 80 nm, 85 nm, 90 nm, 93 nm, 95.44 nm, 98 nm, etc.

[0069] According to an embodiment of the present disclosure, the Zeta potential of the lung-targeted nanomicelles is -21.49 to +20.91 mV, for example, it can be -21.49 mV, -15 mV, -10 mV, 4.7 mV, 10 mV, 20.91 mV, etc.

[0070] As a second aspect of the present disclosure, a method for preparing the above-mentioned lung-targeted nanomicelles is provided, wherein the lung-targeted nanomicelles are prepared by a thin film dispersion method using a permanent cationic lipid, a pH-responsive lipid, and a cationic lipid modified with phenylboronic acid in a desired ratio, and the preparation steps include:

[0071] The permanent cationic lipid, the pH-responsive lipid, and the cationic lipid modified with phenylboronic acid are mixed and dissolved in an organic solvent to obtain a mixed solution;

[0072] removing the organic solvent from the mixed solution to form a lipid film, and drying the lipid film to obtain a dried lipid film;

[0073] The dried lipid film was hydrated with ultrapure water to obtain a lipid suspension, which was then subjected to water bath ultrasonic treatment to form lung-targeted nanomicelles.

[0074] According to an embodiment of the present disclosure, the organic solvent includes at least one of methanol and chloroform, which is used to disperse and dissolve the liposomes to allow them to be fully mixed.

[0075] According to an embodiment of the present disclosure, the method for removing the organic solvent is vacuum rotary evaporation, and the rotation speed of the vacuum rotary evaporation is 80 to 150 rpm, for example, it can be 80 rpm, 100 rpm, 120 rpm, 150 rpm, etc.; the temperature of the vacuum rotary evaporation is 30 to 60°C, for example, it can be 30°C, 40°C, 50°C, 60°C, etc.

[0076] According to an embodiment of the present disclosure, the vacuum drying temperature is 20-30°C, for example, it can be 20°C, 22°C, 25°C, 28°C, 30°C, etc.; the vacuum drying time is 1.5-5h, for example, it can be 1.5h, 2h, 2.5h, 3h, 5h, etc.

[0077] According to an embodiment of the present disclosure, the frequency of ultrasonic treatment is 30-60 kHz, for example, 30 kHz, 40 kHz, 50 kHz, 60 kHz, etc., and the time of ultrasonic treatment is 1-10 min, for example, 1 min, 3 min, 5 min, 7 min, 10 min, etc.

[0078] According to an embodiment of the present disclosure, the steps for preparing lung-targeted nanomicelles by a thin film dispersion method are as follows: permanent cationic lipids, pH-responsive lipids, and cationic lipids modified with phenylboronic acid are weighed according to different molar ratios, mixed in a glass bottle, and methanol is added to fully dissolve them; the methanol solvent is removed by a rotary evaporator at 100 rpm and 40°C under reduced pressure conditions, and after a uniform thin film is formed at the bottom of the glass bottle, the glass bottle is removed and placed in a vacuum drying oven for 2 hours to completely remove the methanol; 1 mL of ultrapure water is added to the dried lipid film to obtain a lipid suspension, and the lipid suspension is ultrasonically treated for about 3 minutes to finally obtain lung-targeted nanomicelles.

[0079] As a third aspect of the present disclosure, a pharmaceutical composition is provided, comprising the aforementioned lung-targeted nanomicelles as drug carriers. As drug delivery vehicles, the lung-targeted nanomicelles can adsorb vitronectin from serum, circulate through the blood, and then specifically bind to the highly expressed αvβ3 cognate receptor in the lungs, thereby increasing drug accumulation and retention time in the lungs, thereby achieving lung targeting.

[0080] As a fourth aspect of the present disclosure, there is provided a use of the above-mentioned lung-targeted nanomicelles in the preparation of a drug for treating lung diseases.

[0081] According to an embodiment of the present disclosure, the lung disease is pneumonia.

[0082] According to the embodiments of the present disclosure, a lung-targeted nanomicelle is constructed, which is composed of a permanent cationic lipid, a pH-responsive lipid, and a cationic lipid modified with phenylboronic acid. By adjusting the composition of the liposomes, especially the ratio of cationic lipids, the surface properties of the nanomicelles are regulated. As the ratio of cationic lipids changes, the surface charge of the nanomicelles changes from negative to positive, increasing the adsorption of targeted lung microenvironment proteins, thereby enabling targeted delivery to the lungs, reducing the dosage of drugs used, and effectively reducing the toxic side effects of drug treatment.

[0083] In order to make the purpose, technical solutions and advantages of the present disclosure more clear and explicit, the technical solutions and principles of the present disclosure are further explained below through specific embodiments in combination with the accompanying drawings. It should be noted that the following specific embodiments are only for illustration and the scope of protection of the present disclosure is not limited thereto.

[0084] Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the examples, these are conventional methods and can be performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0085] The reagents used in the examples were purchased from Sinopharm Chemical Reagent Co., Ltd. and Shanghai Beyotime Biotechnology Co., Ltd., and the C57BL / 6 mice used in the examples were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0086] Example 1

[0087] Preparation of permanent cationic lipids: 5 g of N-tert-butyloxycarbonyl-1,2-ethylenediamine compound was dissolved in anhydrous ethyl acetate, followed by the addition of 17.25 g, 125 mmol, of potassium carbonate (K2CO3) and 38.125 g, 125 mmol, of 1-bromohexadecane (C 16 H 33 The reaction mixture was refluxed and the reaction progress was monitored by thin-layer chromatography. After 48 hours, the mixture was cooled and filtered to remove residual potassium carbonate. After dilution with 200 ml of ethyl acetate, the mixture was washed three times with 200 ml of water, 20 ml of saturated brine was added, and the mixture was dried over anhydrous sodium sulfate. The crude product was evaporated under reduced pressure and purified by column chromatography using 1% MeOH / CHCl₃ (v / v) as the eluent to obtain tert-butyl 2-(hexacosylamino)carbamic acid ethyl ester.

[0088] Then 2g, 3.28mmol of tert-butyl 2-(hexadecylamino) ethyl carbamate was dissolved in 20ml of methyl iodide, 2.0099g, 14.542mmol of potassium carbonate was added, and the reaction mixture was stirred at room temperature for 12 hours to obtain a suspension. The reaction mixture was filtered, and the solvent was evaporated on a rotary evaporator. Then, ether was used for precipitation to obtain hexadecyl N-methyl hexadecyl-1-amine. 2g of hexadecyl N-methyl hexadecyl-1-amine was dissolved in anhydrous dichloromethane, and 5ml of trifluoroacetic acid (TFA) was added dropwise on ice under nitrogen. The reaction mixture was gradually heated to room temperature and stirred for 4h to ensure that the tert-butyl carbonyl (BOC) was completely deprotected. After adding 50ml of chloroform repeatedly 5 times, the mixture was concentrated on a rotary evaporator to completely remove trifluoroacetic acid (TFA). The residual solvent was removed under high vacuum to obtain a permanent cationic lipid.

[0089] FIG1 is a schematic diagram of the synthesis of permanent cationic lipids and pH-responsive lipids in Example 1 of the present disclosure.

[0090] Preparation of pH-responsive lipids: 2.506 g of tert-butyl 2-(hexadecylamino)ethyl carbamate obtained in the above preparation process was taken, 4.115 mmol was dissolved in anhydrous dichloromethane, 5 ml of trifluoroacetic acid (TFA) was added dropwise under nitrogen on ice, and the reaction mixture was gradually heated to room temperature. Stir for 4 hours to ensure that the tert-butyl carbonyl (BOC) was completely deprotected, 50 ml of chloroform was added 5 times, and then concentrated on a rotary evaporator to completely remove the trifluoroacetic acid (TFA) in the solution. The residual solvent was removed under high vacuum, the product was dissolved in methanol, and an equivalent amount of maleic anhydride was added to react to obtain a pH-responsive lipid.

[0091] FIG2 is a schematic diagram of the synthesis of a cationic lipid modified with phenylboronic acid in Example 1 of the present disclosure.

[0092] Preparation of cationic lipids modified with phenylboronic acid: Take another flask and add 1.0g, 16.2mmol 2-aminoethanol dissolved in ethyl acetate, then add 15g, 49mmol 1-bromohexadecane and 2.76g, 16.2mmol potassium carbonate, reflux at a temperature of 85°C for 48h, and the reflux speed is 480rpm. Use an ordinary funnel to filter the potassium carbonate, and after vacuum evaporation to remove the solvent, the residue is extracted with chloroform and washed twice with 50ml water to separate the layers. Dry the lower organic layer with anhydrous sodium sulfate for 2 hours, filter and rotary evaporate the solvent to obtain the product 2-hexadecylethanol. Mix the product 2-hexadecylethanol with 4-(bromomethyl)phenylboronic acid in a molar concentration ratio of 1.5:1 in N,N-dimethylformamide and stir at 60°C for 24 hours. Then precipitate and dry in ether to obtain a cationic lipid modified with phenylboronic acid

[0093] Figure 3 is an electrospray ionization mass spectrometry (ESI-MS) graph of the permanent cationic lipid in Example 1 of the present disclosure, Figure 4 is an electrospray ionization mass spectrometry (ESI-MS) graph of the pH-responsive lipid in Example 1 of the present disclosure, and Figure 5 is an electrospray ionization mass spectrometry (ESI-MS) graph of the phenylboronic acid-modified cationic lipid in Example 1 of the present disclosure. As can be seen from Figures 3, 4, and 5, the molecular weights of the permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid prepared in the present disclosure examples are 523.59 g / mol, 607.57 g / mol, and 644.61 g / mol, respectively.

[0094] Example 2

[0095] The permanent cationic lipid prepared in Example 1 of the present disclosure is used pH-responsive lipids and phenylboronic acid-modified cationic lipids Lung-targeted nanomicelles were prepared by thin film dispersion method, dissolved in methanol solution according to the following 8 different proportions, and mixed in a glass bottle.

[0096] (1) The molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 0:3:1 (the molar content of permanent cationic lipid is 0%);

[0097] (2) the molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 0.5:2.5:1 (the molar content of permanent cationic lipid is 12.5%);

[0098] (3) the molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 1:2:1 (the molar content of permanent cationic lipid is 25%);

[0099] (4) the molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 1.5:1.5:1 (the molar content of permanent cationic lipid is 37.5%);

[0100] (5) The molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 2:1:1 (the molar content of permanent cationic lipid is 50%);

[0101] (6) The molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 2.5:0.5:1 (the molar content of permanent cationic lipid is 62.5%);

[0102] (7) The molar ratio of permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 3:0:1 (the molar content of permanent cationic lipid is 75%);

[0103] (8) The molar ratio of the permanent cationic lipid, pH-responsive lipid, and phenylboronic acid-modified cationic lipid is 4:0:0 (the molar content of the permanent cationic lipid is 100%).

[0104] The organic solvent was removed using a rotary evaporator at 100 rpm and 40°C under reduced pressure conditions, forming a uniform thin film at the bottom of the glass bottle. The glass bottle was removed and placed in a vacuum drying oven for 2 hours to completely remove the methanol. 1 mL of ultrapure water was then added to the dried lipid film to obtain a lipid suspension through hydration reaction. The lipid suspension was then subjected to water bath ultrasonic treatment for approximately 3 minutes to form different lung-targeted nanomicelles.

[0105] The prepared different lung-targeted nanomicelle suspensions were appropriately diluted with ultrapure water, and the particle size distribution and Zeta potential of the lung-targeted nanomicelles were measured at 25°C using a 90Plus PALS high-sensitivity Zeta potential and particle size analyzer.

[0106] Figure 6 is a graph showing the particle size of the lung-targeted nanomicelles in Example 2 of the present disclosure, and Figure 7 is a graph showing the zeta potential of the lung-targeted nanomicelles in Example 2 of the present disclosure. The graph shows that the average particle size of the lung-targeted nanomicelles prepared in Example 2 of the present disclosure is 95.44±17.26 nm, and that the zeta potential of the lung-targeted nanomicelles varies from -21.49 to +20.91 mV as the proportion of permanent cationic lipid added increases (the molar ratio of permanent cationic lipid increases from 0 to 100%).

[0107] Example 3

[0108] When the molar ratio of the permanent cationic lipid, pH-responsive lipid, and cationic lipid modified with phenylboronic acid in Example 2 is 2:1:1 (the molar content of the permanent cationic lipid is 50%), the prepared lung-targeted nanomicelles are dropped one drop onto the carbon film copper mesh with a pipette, and allowed to stand for 1 minute to allow the sample to adsorb onto the carbon film copper mesh. The excess solution is absorbed with filter paper, and the sample is stained with a 7% uranyl acetate aqueous solution for 2 minutes and dried in an oven. The morphological characteristics of the lung-targeted nanomicelles are observed using a transmission electron microscope HT7650 at an accelerating voltage of 100 kV. Figure 8 is a transmission electron microscope (TEM) image of the lung-targeted nanomicelles in Example 3 of the present disclosure.

[0109] The lung-targeted nanomicelles were placed at 4°C for 7 days. Figures 9 and 10 are respectively the particle size stability test graphs and Zeta potential stability test graphs of the lung-targeted nanomicelles in Example 3 of the present disclosure. It can be seen from the figures that the particle size distribution and potential of the lung-targeted nanomicelles did not change significantly after being placed at 4°C for 7 days, and the nanomicelles have high stability.

[0110] Example 4

[0111] Permanent cationic lipids, pH-responsive lipids, and phenylboronic acid-modified cationic lipids were weighed at a molar ratio of 2:1:1, dissolved in methanol, and mixed thoroughly in a glass bottle. A 25 μl solution of 1 mM DiD fluorescent marker dissolved in ethanol was added to the glass bottle. The organic solvent was removed using a rotary evaporator at 100 rpm and 40°C under reduced pressure. The glass bottle was removed and placed in a vacuum drying oven for 2 hours to completely remove the organic solvent. 1 mL of ultrapure water was then added to the dried lipid film. The resulting lipid suspension was sonicated in a water bath for approximately 3 minutes and centrifuged at 2000 rpm for 20 minutes to remove free DiD. The supernatant was collected to obtain DiD fluorescently labeled lung-targeting nanomicelles.

[0112] DiD fluorescently labeled lung-targeted nanomicelles were injected into C57BL / 6 healthy mice via the tail vein. The mice were killed by cervical dislocation 6 hours after administration, and the heart, liver, spleen, lungs, kidneys and other organs were dissected out. The fluorescence intensity was detected by imaging using IVIS Spectrum small animal living imager, and the obtained images were analyzed using Living Imaging software. Figure 11 is an imaging distribution diagram of the fluorescently labeled lung-targeted nanomicelles in Example 4 of the present disclosure. It can be seen from the in vitro fluorescence images in Figure 11 that the nanomicelles with different cationic lipid molar ratios labeled with DiD fluorescent dye are imaged and distributed in mice, including fluorescence imaging of the heart, liver, spleen, lungs, kidneys and other organs of the dissected mice. Among them, the nanomicelles exhibit obvious lung-targeting properties when the permanent cationic lipid molar ratio is 50%.

[0113] DiD fluorescently labeled lung-targeted nanomicelles were injected into mice via the tail vein. Six hours later, the organs were dissected and imaged using an IVIS Spectrum small animal in vivo imager to measure fluorescence intensity, ensuring that the DiD-labeled nanomicelles were in the mice. Lung cryosections were then processed and the fluorescence distribution of the DiD-labeled nanomicelles in the lung tissue was directly observed using a laser confocal scanning microscope (LSM880+Airyscan).

[0114] Figure 12 is a confocal laser scanning microscope (CLSM) image of the DiD fluorescently labeled lung-targeted nanomicelles in Example 4 of the present disclosure. In Figure 12, DiD represents DiD fluorescently labeled lung-targeted nanomicelles, and FITC-CD31 represents lung endothelial cells. In the merged figure, the DiD fluorescently labeled lung-targeted nanomicelles can be seen in the lung parenchyma at the point indicated by the arrow.

[0115] The lung-targeted nanomicelles, preparation methods, and applications provided by the present invention regulate the surface charge properties of the nanomicelles by adjusting the ratio of three lipids: permanent cationic lipids, pH-responsive lipids, and phenylboronic acid-modified cationic lipids. Mouse organ fluorescence imaging, laser confocal microscopy, and other technical means confirm that the lung-targeted nanomicelles can specifically achieve lung-targeted delivery, thereby providing a new means for achieving efficient treatment of pneumonia and a potential opportunity for achieving targeted delivery of drugs in lung tissue. The drug dosage can be reduced, and the toxic side effects of drug treatment on other tissues can be effectively reduced, showing great clinical application prospects.

[0116] The specific embodiments of the present disclosure described above do not limit the scope of protection of the present disclosure. Any other corresponding changes and modifications made based on the technical concept of the present disclosure should be included in the scope of protection of the claims of the present disclosure.

Claims

1. A lung-targeted nanomicelle, comprising: Permanent cationic lipids; pH-responsive lipids; as well as Phenylboronic acid-modified cationic lipids; The molar ratio of the permanent cationic lipid, the pH-responsive lipid, and the cationic lipid modified by phenylboronic acid is X:3-X:1, wherein X=1-2, so that the nanomicelles are regulated to target the lungs.

2. The nano micelle according to claim 1, wherein The permanent cationic lipid has a structure shown in Formula I: Wherein, R1 is a C12-C18 alkyl group.

3. The nano micelle according to claim 1, wherein The pH responsive lipid has a structure shown in Formula II: Wherein, R2 is a C12-C18 alkyl group.

4. The nano micelle according to claim 1, wherein The phenylboronic acid-modified cationic lipid has a structure shown in Formula III: Wherein, R3 is a C12-C18 alkyl group.

5. The nano micelle according to claim 1, wherein The average particle size of the lung-targeted nanomicelles is 95.44±17.26 nm.

6. The nano micelle according to claim 1, wherein The zeta potential of the lung-targeted nanomicelles is -21.49 to +20.91 mV.

7. A method for preparing the lung-targeted nanomicelles according to any one of claims 1 to 6, comprising: The permanent cationic lipid, the pH-responsive lipid, and the cationic lipid modified with phenylboronic acid are mixed and dissolved in an organic solvent to obtain a mixed solution; removing the organic solvent in the mixed solution to form a lipid film, and drying the lipid film to obtain a dried lipid film; The dried lipid film is hydrated with ultrapure water to obtain a lipid suspension, and then the lipid suspension is subjected to water bath ultrasonic treatment to form the lung-targeted nano-micelles.

8. The method according to claim 7, wherein: The organic solvent includes at least one of methanol and chloroform.

9. The method according to claim 7, wherein: The method for removing the organic solvent is vacuum rotary evaporation, the speed of the vacuum rotary evaporation is 80-150 rpm, and the temperature of the vacuum rotary evaporation is 30-60° C.; The vacuum drying temperature is 20-30°C, and the vacuum drying time is 1.5-5h; The frequency of the ultrasonic treatment is 30-60 kHz, and the time of the ultrasonic treatment is 1-10 minutes.

10. A pharmaceutical composition comprising the lung-targeting nanomicelle according to any one of claims 1 to 9 as a drug carrier.

11. Use of the lung-targeted nanomicelle according to any one of claims 1 to 9 in the preparation of a drug for treating lung diseases.

12. The use according to claim 11, wherein: The lung disease is pneumonia.

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