WO2024250133A1 - Lung-targeted nano-micelle, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a lung-targeted nano-micelle, and a preparation method therefor and the use thereof, wherein the lung-targeted nano-micelle comprises: a permanent cationic lipid, a pH responsive lipid, and a phenylboronic acid-modified cationic lipid. The molar ratio of the permanent cationic lipid, the pH responsive lipid and the phenylboronic acid-modified cationic lipid is X:(3-X):1, wherein X=1-2, such that the nano-micelle is regulated to target a lung.

Description

Lung-targeted nanomicelles, preparation methods and applications Technical Field

The present invention belongs to the technical field of biomedicine and molecular biology, and relates to a lung-targeted nano-micelle, a preparation method and an application thereof.

Background Art

Lung disease is one of the common diseases that threaten human life and health. Bacterial pneumonia is a common lung disease. Nebulized and oral administration have poor patient compliance and poor treatment effects. In addition, intravenous antibiotics are a relatively effective treatment method. However, the emergence of multidrug-resistant bacteria and the poor targeting, low utilization rate, and short lung residence time of antibiotic treatment face problems such as inability to achieve lung targeting, resulting in poor treatment effects. Frequent use of antibiotics at high doses can lead to drug resistance and a series of toxic side effects, such as hepatotoxicity and renal toxicity and intestinal flora imbalance. Therefore, the development of a nano-micelle that can achieve lung targeting has important clinical practical significance in the treatment of lung diseases.

Summary of the invention

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

In order to solve the above technical problems, the technical solutions provided by the present disclosure are as follows:

As a first aspect of the present disclosure, a lung-targeted nano-micelle is provided, comprising:

Permanent cationic lipids;

pH responsive lipids; and

Phenylboronic acid-modified cationic lipids;

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

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

Wherein, _R1 is a C12-C18 alkyl group.

In one embodiment, the pH responsive lipid has a structure shown in Formula II:

Wherein, _R2 is a C12-C18 alkyl group.

In one embodiment, the cationic lipid modified with phenylboronic acid has a structure shown in Formula III:

Wherein, _R3 is a C12-C18 alkyl group.

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

In one embodiment, the zeta potential of the lung-targeted nanomicelles is -21.49 to +20.91 mV.

As a second aspect of the present disclosure, a method for preparing the above-mentioned lung-targeted nanomicelles is provided, 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 was hydrated with ultrapure water to obtain a lipid suspension, which was then subjected to water bath sonication to form lung-targeted nanomicelles.

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

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.;

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

The frequency of ultrasonic treatment is 30-60 kHz, and the time of ultrasonic treatment is 1-10 min.

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

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.

In one embodiment, the lung disease is pneumonia.

The lung-targeted nanomicelles, preparation methods and applications provided by the present disclosure are constructed from three lipids: permanent cationic lipids, pH-responsive lipids and cationic lipids modified with phenylboronic acid. The surface charge properties of the nanomicelles can be regulated by adjusting the ratio of the permanent cationic lipids. As the ratio of the permanent cationic lipids changes, the surface charge of the nanomicelles changes from negative to positive, which can increase the adsorption of targeted lung microenvironment proteins and achieve targeted delivery to the lungs. It can be used in the respiratory system, providing a potential opportunity for achieving targeted delivery of drugs in lung tissue, reducing the dosage of drugs, and effectively reducing the toxic and side effects of drug treatment on other tissues, showing a large clinical application prospect.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

FIG7 is a Zeta potential diagram of the lung-targeted nanomicelles in Example 2 of the present disclosure;

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

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

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

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

FIG. 12 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

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 combination with specific embodiments and with reference to the accompanying drawings.

At present, liposomes with amphiphilic structures are widely used in drug carriers. Due to their good designability, they can well target delivery and continuously release loaded drugs, enhance the toxic side effects of drugs, and optimize their pharmacokinetics. However, after entering the blood, traditional liposomes will absorb a large amount of natural immunoglobulin M (IgM), which intensifies the body's immune system's recognition of liposomes and enriches them in the liver and spleen. Therefore, most of the drugs transported by liposomes are mainly enriched in the liver and spleen, and cannot be effectively delivered to the target organs.

In the process of realizing the present disclosure, it was found 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 lung. 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.

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

Permanent cationic lipids;

pH responsive lipids; and

Phenylboronic acid-modified cationic lipids;

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

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 charges on proteins in the blood; pH-responsive lipids are liposomes with pH-responsive functions, 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 lipids in unfolding. The molar ratio of the permanent cationic lipids, pH-responsive lipids, and phenylboronic acid-modified cationic lipids 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 cationic lipids modified by phenylboronic acid remains unchanged. When the molar ratio of the permanent cationic lipids, pH-responsive lipids, and phenylboronic acid-modified cationic lipids is 2:1:1, the surface charge of the obtained nanomicelles is neutral to positive, which is conducive to the adsorption of vitronectin in serum. After reaching the lungs through the blood circulation, it specifically binds to the αvβ3 homologous receptors highly expressed in the lungs, thereby increasing the enrichment and retention time of the drug in the lungs, thereby completing lung targeting.

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

Wherein, _R1 is a C12-C18 alkyl group, which is used to regulate the charge, thereby regulating the adsorption of the nanomicelles to proteins in the blood, so that the nanomicelles are regulated to be lung-targeted.

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

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.

Reaction with methyl halides to obtain products Wherein, _R1 is a C12-C18 alkyl group.

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.

According to an embodiment of the present disclosure, the pH responsive lipid has a structure shown in Formula II:

Among them, R ₂ is a C12-C18 alkyl group, which carries a negative charge. The liposomes with pH response function can achieve bacterial targeting through amide bonds in a slightly acidic bacterial environment.

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

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.

Take it off again tert-butyl carbonyl (BOC), add maleic anhydride to react to obtain pH Responsive lipids Wherein, _R2 is a C12-C18 alkyl group.

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

Wherein, R ₃ is an alkyl group of C12 to C18. The cationic lipid modified with phenylboronic acid has a neutral charge, and the covalent bond between the boronic acid group and the bacterial polysaccharide cis-diol is used to achieve precise targeting of the bacterial infection microregion in the lung. The cationic lipid modified with phenylboronic acid that responds to active oxygen and the pH-responsive lipid can assist the pH-responsive lipid in unfolding its action, and further utilize the micro-acid and active oxygen stimulation of the bacterial infection microregion to transform the nanomicelle structure and quickly release the loaded antibiotics.

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

2-aminoethanol and C12-C18 alkyl bromide are reacted under the action of an alkali catalyst to obtain the product alkyl ethanol. Wherein, _R3 is a C12-C18 alkyl group.

Then Reaction with 4-(bromomethyl)phenylboronic acid to obtain phenylboronic acid-modified cationic lipids Wherein, _R3 is a C12-C18 alkyl group.

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.

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.

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

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 was hydrated with ultrapure water to obtain a lipid suspension, which was then subjected to water bath sonication to form lung-targeted nanomicelles.

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.

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.

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.

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

According to an embodiment of the present disclosure, the steps of 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.

As a third aspect of the present disclosure, a pharmaceutical composition is provided, comprising the above-mentioned lung-targeted nano-micelles as a drug carrier. The above-mentioned lung-targeted nano-micelles as drug delivery carriers can absorb vitronectin in serum and then be transported to the blood circulation. After reaching the lungs, it specifically binds to the αvβ3 homologous receptors that are highly expressed in the lungs, increasing the drug's accumulation and retention time in the lungs, thereby achieving lung targeting.

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.

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

According to the embodiments of the present disclosure, the present disclosure constructs a lung-targeted nano-micelle, which is composed of permanent cationic lipids, pH-responsive lipids and cationic lipids modified with phenylboronic acid, and the surface properties of the nano-micelles are regulated by adjusting the composition of the liposomes, especially the proportion of the cationic liposomes. As the proportion of cationic lipids changes, the surface charge of the nano-micelles changes from negative to positive, which increases the adsorption of targeted lung microenvironment proteins, and then can achieve targeted delivery to the lungs, which can reduce the dosage of the drug and effectively reduce the toxic side effects of drug treatment.

In order to make the purpose, technical solution and advantages of the present disclosure clearer and more specific, the technical solution and principle of the present disclosure are further explained below through specific embodiments combined with the accompanying drawings. It should be noted that the following specific embodiments are only used as examples, and the protection scope of the present disclosure is not limited thereto.

The test materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Specific techniques or conditions not specified in the examples are conventional methods and can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

The reagents used in the examples were purchased from Sinopharm Chemical Reagent Co., Ltd. and Shanghai Bio-Tech Biotechnology Co., Ltd., and the C57BL/6 mice used in the examples were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Example 1

Preparation of permanent cationic lipids: 5 g of N-tert-butyloxycarbonyl-1,2-ethylenediamine compound was dissolved in anhydrous ethyl acetate, followed by addition of 17.25 g, 125 mmol of potassium carbonate (K ₂ CO ₃ ) and 38.125 g, 125 mmol of 1-bromohexadecane (C ₁₆ H ₃₃ Br). The reaction mixture was refluxed and the reaction process was monitored by thin layer chromatography. After 48 h, the mixture was cooled and filtered to remove potassium carbonate residues. After dilution with 200 ml of ethyl acetate, the mixture was washed with 200 ml of water for 3 times, 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/CHCl ₃ (v/v) as the eluent to obtain the product tert-butyl 2-(hexacosylamino)ethyl carbamate.

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 suspension was obtained by stirring at room temperature for 12 hours. The reaction mixture was filtered, and the solvent was evaporated on a rotary evaporator, and then precipitated with ether 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, it was concentrated on a rotary evaporator to completely remove trifluoroacetic acid (TFA). The residual solvent was removed under high vacuum to obtain permanent cationic lipids

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

Preparation of pH-responsive lipids: Take 2.506 g of tert-butyl 2-(hexadecylamino)ethyl carbamate obtained in the above preparation process, dissolve 4.115 mmol in anhydrous dichloromethane, drop 5 ml of trifluoroacetic acid (TFA) under nitrogen on ice, and gradually heat the reaction mixture to room temperature. Stir for 4 hours to ensure that the tert-butyl carbonyl (BOC) is completely deprotected, repeat the addition of 50 ml of chloroform 5 times, and then concentrate on a rotary evaporator to completely remove trifluoroacetic acid (TFA) in the solution. Remove the residual solvent under high vacuum, dissolve the product in methanol, and add an equivalent amount of maleic anhydride to react to obtain pH-responsive lipids.

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

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 85°C for 48h, and the reflux speed is 480rpm. Use an ordinary funnel to filter the potassium carbonate, and evaporate the solvent under reduced pressure to remove the solvent. Then extract the residue with chloroform and wash it with 50ml water twice to separate the layers. Dry the lower organic layer with anhydrous sodium sulfate for 2 hours, filter and 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

FIG3 is an electrospray ionization mass spectrometry (ESI-MS) diagram of the permanent cationic lipid in Example 1 of the present disclosure, FIG4 is an electrospray ionization mass spectrometry (ESI-MS) diagram of the pH-responsive lipid in Example 1 of the present disclosure, and 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. It can be seen from FIG3, FIG4, and FIG5 that the molecules of the permanent cationic lipid, pH-responsive lipid, and cationic lipid modified with phenylboronic acid prepared in the present disclosure example The amounts are 523.59 g/mol, 607.57 g/mol and 644.61 g/mol respectively.

Example 2

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.

(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%);

(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%);

(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%);

(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%);

(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%);

(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%);

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

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

The organic solvent was removed using a rotary evaporator at 100 rpm and 40°C under reduced pressure conditions, and a uniform thin film was formed 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. Then 1 mL of ultrapure water was added to the dried lipid film to obtain a lipid suspension through hydration reaction. The lipid suspension was treated with water bath ultrasound for about 3 minutes to form different lung-targeted nanomicelles.

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 using a 90Plus PALS high-sensitivity Zeta potential and particle size analyzer at 25°C.

Figure 6 is a particle size diagram of the lung-targeted nanomicelles in Example 2 of the present disclosure, and Figure 7 is a Zeta potential diagram of the lung-targeted nanomicelles in Example 2 of the present disclosure. It can be seen from the figure that the average particle size of the lung-targeted nanomicelles prepared in the present disclosure example is 95.44±17.26nm, and as the addition ratio of the permanent cationic lipid increases (the molar ratio of the permanent cationic lipid increases from 0 to 100%), the Zeta potential of the lung-targeted nanomicelles changes from -21.49 to +20.91mV.

Example 3

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 1 drop on the carbon film copper mesh with a pipette, left to stand for 1 minute to allow the sample to adsorb to the carbon film copper mesh, and the excess solution is absorbed with filter paper, and the sample is stained with 7% uranyl acetate aqueous solution for 2 minutes, dried in an oven, and the morphological characteristics of the lung-targeted nanomicelles are observed with 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.

The lung-targeted nanomicelles were placed at 4°C for 7 days. Figures 9 and 10 are respectively the particle size stability test graph and Zeta potential stability test graph 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.

Example 4

Permanent cationic lipids, pH-responsive lipids, and cationic lipids modified with phenylboronic acid were weighed at a molar ratio of 2:1:1, dissolved in methanol, and then mixed evenly in a glass bottle. 25ul of DiD fluorescence dissolved in ethanol at a concentration of 1mM was mixed into a glass bottle, and the organic solvent was removed using a rotary evaporator at 100rpm and 40℃ under reduced pressure conditions. The glass bottle was removed and placed in a vacuum drying oven for 2 hours to completely remove the organic solvent, and 1mL of ultrapure water was added to the dried lipid film. The lipid suspension obtained was subjected to water bath ultrasonic treatment for about 3 minutes, and low-speed centrifugation at 2000rpm for 20min was performed to remove free DiD, and the supernatant was taken to obtain DiD fluorescently labeled lung-targeted nanomicelles.

DiD fluorescently labeled lung-targeted nanomicelles were injected into C57BL/6 healthy mice via the tail vein. After 6 hours, the mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney and other organs were dissected out. The fluorescence intensity was detected by imaging using IVIS Spectrum small animal living imager, and the obtained pictures 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 image in Figure 11 that the nanomicelles with different cationic lipid molar ratios labeled with DiD fluorescent dye are imaged and distributed in mice, including the fluorescence imaging of the heart, liver, spleen, lung, kidney and other organs of the dissected mice, among which, the permanent cationic lipid molar ratio in the nanomicelles is 50%, which shows obvious lung-targeting properties.

DiD fluorescently labeled lung-targeted nanomicelles were injected into mice through the tail vein. The organs were dissected out 6 hours later, and then the fluorescence intensity was detected by imaging using IVIS Spectrum small animal in vivo imager to ensure that the DiD-labeled nanomicelles were in the mice. Lung frozen sections were processed and the fluorescence distribution of DiD-labeled nanomicelles in lung tissue was directly observed using a laser confocal scanning microscope (LSM880+Airyscan).

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

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, namely, permanent cationic lipids, pH-responsive lipids and cationic lipids modified with phenylboronic acid. Mouse organ fluorescence imaging, laser confocal microscopy and other technical means are used to 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, which can reduce the dosage of drugs used and effectively reduce the toxic and side effects of drug treatment on other tissues, showing great clinical application prospects.

The specific implementations of the present disclosure described above do not constitute a limitation on the protection scope of the present disclosure. Any other corresponding changes and modifications made according to the technical concept of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Claims (12)

A lung-targeted nano-micelle comprising: Permanent cationic lipids; pH responsive lipids; and 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. The nanomicelle according to claim 1, wherein The permanent cationic lipid has a structure shown in Formula I: Wherein, _R1 is a C12-C18 alkyl group. The nanomicelle according to claim 1, wherein The pH responsive lipid has a structure shown in Formula II: Wherein, _R2 is a C12-C18 alkyl group. The nanomicelle 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. The nanomicelle according to claim 1, wherein The average particle size of the lung-targeted nanomicelles is 95.44±17.26 nm. The nanomicelle according to claim 1, wherein The zeta potential of the lung-targeted nanomicelles is -21.49 to +20.91 mV. 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. The method according to claim 7, wherein: The organic solvent includes at least one of methanol and chloroform. 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. A pharmaceutical composition comprising the lung-targeting nanomicelle according to any one of claims 1 to 9 as a drug carrier. A use of the lung-targeted nanomicelles as claimed in any one of claims 1 to 9 in the preparation of a drug for treating lung diseases. The use according to claim 11, wherein The lung disease is pneumonia.