WO2024250133A1 - 肺靶向纳米胶束、制备方法及应用 - Google Patents

肺靶向纳米胶束、制备方法及应用 Download PDF

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WO2024250133A1
WO2024250133A1 PCT/CN2023/098249 CN2023098249W WO2024250133A1 WO 2024250133 A1 WO2024250133 A1 WO 2024250133A1 CN 2023098249 W CN2023098249 W CN 2023098249W WO 2024250133 A1 WO2024250133 A1 WO 2024250133A1
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lung
lipid
targeted
nanomicelles
responsive
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French (fr)
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丁圣刚
傅玲
霍绍虎
聂旋
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First Affiliated Hospital of Anhui Medical University
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First Affiliated Hospital of Anhui Medical University
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Priority to PCT/CN2023/098249 priority patent/WO2024250133A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton

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  • 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.
  • 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.
  • intravenous antibiotics are a relatively effective treatment method.
  • 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.
  • 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.
  • a lung-targeted nano-micelle comprising:
  • Phenylboronic acid-modified cationic lipids Phenylboronic acid-modified cationic lipids
  • the permanent cationic lipid has the structure shown in Formula I:
  • R1 is a C12-C18 alkyl group.
  • the pH responsive lipid has a structure shown in Formula II:
  • R2 is a C12-C18 alkyl group.
  • the cationic lipid modified with phenylboronic acid has a structure shown in Formula III:
  • 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;
  • 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
  • FIG10 is a graph showing the Zeta potential stability test of the lung-targeted nanomicelles in Example 3 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.
  • CLSM confocal laser scanning microscope
  • 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.
  • the present disclosure provides a lung-targeted nanomicelle composed of permanent cationic lipids, pH-responsive lipids and phenylboronic acid-modified cationic lipids.
  • a lung-targeted nanomicelle composed of permanent cationic lipids, pH-responsive lipids and phenylboronic acid-modified cationic lipids.
  • Phenylboronic acid-modified cationic lipids Phenylboronic acid-modified cationic lipids
  • 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.
  • 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 ⁇ v ⁇ 3 homologous receptors highly expressed in the lungs, thereby increasing the enrichment and retention time of the drug in the lungs, thereby completing lung targeting.
  • the permanent cationic lipid has a structure shown in Formula I:
  • 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.
  • 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.
  • R1 is a C12-C18 alkyl group.
  • R1 is a C12-C18 alkyl group.
  • the pH responsive lipid has a structure shown in Formula II:
  • 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.
  • the method for preparing pH responsive lipids is as follows:
  • R2 is a C12-C18 alkyl group.
  • R2 is a C12-C18 alkyl group.
  • the cationic lipid modified with phenylboronic acid has a structure shown in Formula III:
  • 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.
  • the method for preparing a phenylboronic acid-modified cationic lipid is as follows:
  • R3 is a C12-C18 alkyl group.
  • R3 is a C12-C18 alkyl group.
  • 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.
  • 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.
  • a method for preparing the above-mentioned lung-targeted nano-micelles 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 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.
  • the method for removing the organic solvent is vacuum rotary evaporation
  • 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.
  • 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.
  • the frequency of ultrasonic treatment is 30-60kHZ, for example, 30kHZ, 40kHZ, 50kHZ, 60kHZ, etc.
  • the time of ultrasonic treatment is 1-10min, for example, 1min, 3min, 5min, 7min, 10min, etc.
  • 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.
  • a pharmaceutical composition 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 ⁇ v ⁇ 3 homologous receptors that are highly expressed in the lungs, increasing the drug's accumulation and retention time in the lungs, thereby achieving lung targeting.
  • the lung disease is pneumonia.
  • test materials and reagents used in the following examples 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.
  • 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.
  • TFA trifluoroacetic acid
  • 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 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
  • 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.
  • 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.
  • 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%);
  • 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.
  • 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
  • Figure 7 is a Zeta potential diagram of the lung-targeted nanomicelles in Example 2 of the present disclosure.
  • the average particle size of the lung-targeted nanomicelles prepared in the present disclosure example is 95.44 ⁇ 17.26nm
  • the Zeta potential of the lung-targeted nanomicelles changes from -21.49 to +20.91mV.
  • 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.
  • FIGS 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.
  • 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°C 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.
  • 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.
  • DiD represents DiD fluorescence-labeled lung-targeted nanomicelles
  • FITC-CD31 represents lung endothelial cells
  • 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.

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Abstract

提供一种肺靶向纳米胶束、制备方法及应用，其中，肺靶向纳米胶束包括：永久性阳离子脂质，pH响应性脂质，以及苯硼酸修饰的阳离子脂质；永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的摩尔比例为X：3-X：1，其中X＝1～2，使得纳米胶束被调控为靶向肺部。

Description

肺靶向纳米胶束、制备方法及应用 技术领域
本公开属于生物医药和分子生物学技术领域，涉及一种肺靶向纳米胶束、制备方法及应用。
背景技术
肺部疾病是威胁人类生命和健康的常见疾病之一，细菌性肺炎是常见肺部疾病，雾化及口服给药患者依从性差，治疗效果欠佳；另外，静脉用抗生素是较为有效的治疗手段，然而多重耐药菌的出现以及抗生素治疗给药面临靶向性差、利用率低、肺部驻留时间短等问题，无法实现肺部靶向，从而导致治疗效果差，且高剂量频繁使用抗生素会导致耐药性产生，并带来一系列毒副作用，如：肝肾毒性和肠道菌群失调等。因此，开发一种实现肺部靶向的纳米胶束在治疗肺部疾病中具有重要临床实际意义。
发明内容
针对上述技术问题，本公开提供了一种肺靶向纳米胶束、制备方法及应用，以期至少部分地解决上述提及的技术问题。
为了解决上述技术问题，本公开提供的技术方案如下：
作为本公开的第一个方面，提供了一种肺靶向纳米胶束，包括：
永久性阳离子脂质；
pH响应性脂质；以及
苯硼酸修饰的阳离子脂质；
其中，永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的摩尔比例为X：3-X：1，其中X＝1～2，使得纳米胶束被调控为靶向肺部。
在其中一个实施例中，永久性阳离子脂质具有式Ⅰ所示结构：
其中，R1为C12～C18的烷基。
在其中一个实施例中，pH响应性脂质具有式II所示结构：
其中，R2为C12～C18的烷基。
在其中一个实施例中，苯硼酸修饰的阳离子脂质具有式III所示结构：
其中，R3为C12～C18的烷基。
在其中一个实施例中，肺靶向纳米胶束的平均粒径为95.44±17.26nm。
在其中一个实施例中，肺靶向纳米胶束的Zeta电位为-21.49～+20.91mV。
作为本公开的第二个方面，提供了一种制备上述的肺靶向纳米胶束的方法，包括：
将永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质混合溶解到有机溶剂中，得到混合液；
除去混合液中的有机溶剂以形成脂质薄膜，并对脂质薄膜进行干燥，得到干燥后的脂质薄膜；
用超纯水水合处理干燥后的脂质薄膜，得到脂质悬浮液后，对脂质悬浮液进行水浴超声处理，形成肺靶向纳米胶束。
在其中一个实施例中，有机溶剂包括甲醇、三氯甲烷中的至少一种。
在其中一个实施例中，除去有机溶剂的方法为减压旋蒸，减压旋蒸的转速为80～150rpm，减压旋蒸的温度为30～60℃；
真空干燥的温度为20～30℃，真空干燥的时间为1.5～5h；
超声处理的频率为30～60kHZ，超声处理的时间为1～10min。
作为本公开的第三个方面，提供了一种药物组合物，包含上述肺靶向纳米胶束，作为药物载体。
作为本公开的第四个方面，提供了一种上述肺靶向纳米胶束在制备用于治疗肺部疾病的药物中的应用。
在其中一个实施例中，肺部疾病为肺炎。
本公开提供的肺靶向纳米胶束、制备方法及应用，由永久性阳离子脂质、pH响应性脂质和苯硼酸修饰的阳离子脂质三种脂质构建肺靶向纳米胶束，通过调节永久性阳离子脂质的比例，可以调控纳米胶束的表面电荷性质，随着永久性阳离子脂质比例的变化，纳米胶束的表面电荷由负到正变化，可以增加对靶向肺部微环境蛋白的吸附，实现针对肺部的靶向递送。可用于呼吸系统，为实现药物在肺组织的靶向递送提供了潜在的机会，减少药物的使用剂量，有效降低药物治疗对其他组织的毒副作用，表现出较大的临床应用前景。
附图说明
图1为本公开实施例1中永久性阳离子脂质和pH响应性脂质的合成示意图；
图2为本公开实施例1中苯硼酸修饰的阳离子脂质的合成示意图；
图3为本公开实施例1中永久性阳离子脂质的电喷雾电离质谱(ESI-MS)图；
图4为本公开实施例1中pH响应性脂质的电喷雾电离质谱(ESI-MS)图；
图5为本公开实施例1中苯硼酸修饰的阳离子脂质的电喷雾电离质谱(ESI-MS)图；
图6为本公开实施例2中肺靶向纳米胶束的粒径图；
图7为本公开实施例2中肺靶向纳米胶束的Zeta电位图；
图8为本公开实施例3中肺靶向纳米胶束的透射电子显微镜(TEM)图；
图9为本公开实施例3中肺靶向纳米胶束的粒径稳定性测试图；
图10为本公开实施例3中肺靶向纳米胶束的Zeta电位稳定性测试图；
图11为本公开实施例4中DiD荧光标记的肺靶向纳米胶束的成像分布图；
图12为本公开实施例4中DiD荧光标记的肺靶向纳米胶束的激光扫描共聚焦显微镜(CLSM)图。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本公开作进一步的详细说明。
目前，具有两亲性结构的脂质体被广泛应用于药物载体中，由于良好的可设计性使其能很好地靶向递送并持续的释放负载药物，增强药物的毒副作用，优化其药代动力学。然而，传统的脂质体进入血液后会大量吸附天然的免疫球蛋白M(IgM)，加剧机体免疫系统对脂质体的识别而富集在肝脾，故而大部分脂质体运输的药物主要富集在肝脾部位，无法有效输送到靶器官。
在实现本公开的过程中发现，相关技术通过调节磷脂、胆固醇、聚乙二醇修饰的脂质体和阳离子脂质体比例可以制备一系列具有不同表面电荷的核酸复合物，调整阳离子脂质体的比例可以实现核酸复合物对不同器官的靶向递送，随着阳离子脂质体比例的变化，运送的荧光素酶能选择性的在肝脏、脾脏与肺部表达。然而，相关技术对肺部靶向递送仅仅出现在基因治疗领域，用于肺部的靶向递送药物鲜有报道。鉴于相关技术中存在的技术问题，本公开提供了一种由永久性阳离子脂质、pH响应性脂质和苯硼酸修饰的阳离子脂质组成的肺靶向纳米胶束，通过调节脂质体的组成比例，尤其是永久性阳离子脂质体在胶束中的比例，能够实现肺部的靶向递送。
为了实现以上技术目标，作为本公开的第一个方面，提供了一种肺靶向纳米胶束，包括：
永久性阳离子脂质；
pH响应性脂质；以及
苯硼酸修饰的阳离子脂质；
其中，永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的摩尔比例为X：3-X：1，其中X＝1～2，使得纳米胶束被调控为靶向肺部。
根据本公开的实施例，永久性阳离子脂质用于调控纳米胶束的表面电荷，从而调控电荷对血液中蛋白的吸附性能；pH响应性脂质为具有pH响应功能的脂质体，可以偏酸性的细菌环境下，以酰胺键实现细菌靶向，可以在弱酸性微环境下实现电荷反转，通过静电相互作用靶向于表面带负电荷的细胞；苯硼酸修饰的阳离子脂质中的二元醇结构具有细菌靶向，辅助pH响应性脂质展开作用。组成纳米胶束的永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的摩尔比例可以是1:2:1、1.2:1.8:1、1.5:1.5:1、1.8:1.2:1、2:1:1等，按照此比例调控纳米胶束为靶向肺部，苯硼酸修饰的阳离子脂质的比例保持不变，当永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的摩尔比例时2:1:1时，得到的纳米胶束表面电荷为中性偏正，有利于吸附血清中的玻连蛋白，经血液循环达到肺部后与肺部高表达的αvβ3同源受体特异性结合，增加药物在肺部的富集和滞留时间，从而完成肺部靶向。
根据本公开的实施例，永久性阳离子脂质具有式Ⅰ所示结构：
其中，R1为C12～C18的烷基，用于调控电荷，从而调控纳米胶束对血液中蛋白的吸附，使纳米胶束被调控为肺部靶向。
根据本公开的实施例，制备永久性阳离子脂质的方法如下：
将N-叔丁氧羰基-1，2-乙二胺和C12～C18的烷基溴在碱催化剂作用下进行反应，得到其中，R1为C12～C18的烷基。
和卤代甲烷反应，得到产物其中，R1为C12～C18的烷基。
再将上述产物的叔丁基碳基(BOC)完全脱保护。得到永久性阳离子脂质其中，R1为C12～C18的烷基。
根据本公开的实施例，pH响应性脂质具有式II所示结构：
其中，R2为C12～C18的烷基，带有负电荷，具有pH响应功能的脂质体，可以偏酸性的细菌环境下，以酰胺键实现细菌靶向。
根据本公开的实施例，制备pH响应性脂质的方法如下：
将叔丁基(2-氨基乙基)氨基甲酸酯化合物和C12～C18的烷基溴在碱催化剂作用下进行反应，得到其中，R2为C12～C18的烷基。
再脱去的叔丁基碳基(BOC)，加入马来酸酐反应得到pH 响应性脂质其中，R2为C12～C18的烷基。
根据本公开的实施例，苯硼酸修饰的阳离子脂质具有式III所示结构：
其中，R3为C12～C18的烷基。苯硼酸修饰的阳离子脂质具有中性电荷，利用硼酸基团与细菌多糖顺式-二醇之间的共价键，实现肺部细菌感染微区的精准靶向，活性氧响应的苯硼酸修饰的阳离子脂质和pH响应性脂质一起，可辅助pH响应性脂质展开作用，进一步利用细菌感染微区的微酸与活性氧刺激作用，使纳米胶束结构发生转变，快速释放出装载的抗生素。
根据本公开的实施例，制备苯硼酸修饰的阳离子脂质的方法如下：
将2-氨基乙醇和C12～C18的烷基溴在碱催化剂作用下进行反应，得到产物烷基乙醇其中，R3为C12～C18的烷基。
再将与4-(溴甲基)苯硼酸反应，得到苯硼酸修饰的阳离子脂质其中，R3为C12～C18的烷基。
根据本公开的实施例，肺靶向纳米胶束的平均粒径为95.44±17.26nm，肺靶向纳米胶束的粒径可以是80nm、85nm、90nm、93nm、95.44nm、98nm等。
根据本公开的实施例，肺靶向纳米胶束的Zeta电位为-21.49～+20.91mV，例如可以是-21.49mV、-15mV、-10mV、4.7mV、10mV、20.91mV等。
作为本公开的第二个方面，提供了一种制备上述的肺靶向纳米胶束的方法，由永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质按照所需比例采用薄膜分散法制备肺靶向纳米胶束，制备步骤包括：
将永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质混合溶解到有机溶剂中，得到混合液；
除去混合液中的有机溶剂以形成脂质薄膜，并对脂质薄膜进行干燥，得到干燥后的脂质薄膜；
用超纯水水合处理干燥后的脂质薄膜，得到脂质悬浮液后，对脂质悬浮液进行水浴超声处理，形成肺靶向纳米胶束。
根据本公开的实施例，有机溶剂包括甲醇、三氯甲烷中的至少一种，用于分散溶解脂质体使其充分混合。
根据本公开的实施例，除去有机溶剂的方法为减压旋蒸，减压旋蒸的转速为80～150rpm，例如可以是80rpm、100rpm、120rpm、150rpm等；减压旋蒸的温度为30～60℃，例如可以是30℃、40℃、50℃、60℃等。
根据本公开的实施例，真空干燥的温度为20～30℃，例如可以是20℃、22℃、25℃、28℃、30℃等；真空干燥的时间为1.5～5h，例如可以是1.5h、2h、2.5h、3h、5h等。
根据本公开的实施例，超声处理的频率为30～60kHZ，例如可以是30kHZ、40kHZ、50kHZ、60kHZ等，超声处理的时间为1～10min，例如可以是1min、3min、5min、7min、10min等。
根据本公开的实施例，采用薄膜分散法制备肺靶向纳米胶束的步骤为：将永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质按照不同的摩尔比例进行称重，在玻璃瓶中混合，加入甲醇使其充分溶解；使用旋转蒸发仪在100rpm和40℃减压条件下去除甲醇溶剂，在玻璃瓶底形成一层均匀薄膜后，将玻璃瓶移出并在真空干燥箱中放置2小时以完全去除甲醇；将1mL超纯水添加到干燥的脂质膜中，得到的脂质悬浮液，对脂质悬浮液超声处理约3分钟，最终得到肺靶向纳米胶束。
作为本公开的第三个方面，提供了一种药物组合物，包含上述肺靶向纳米胶束，作为药物载体。上述肺靶向纳米胶束作为药物递送载体可通过吸附血清中的玻连蛋白，经血液循环 达到肺部后与肺部高表达的αvβ3同源受体特异性结合，增加药物在肺部的富集和滞留时间，从而完成肺部靶向。
作为本公开的第四个方面，提供了一种上述肺靶向纳米胶束在制备用于治疗肺部疾病的药物中的应用。
根据本公开的实施例，肺部疾病为肺炎。
根据本公开的实施例，本公开构建了一种肺靶向纳米胶束，由永久性阳离子脂质、pH响应性脂质和苯硼酸修饰的阳离子脂质组成，通过调节脂质体的组成，尤其是阳离子脂质体的比例，调控纳米胶束的表面性质。随着阳离子脂质比例的变化，纳米胶束的表面电荷由负到正的变化，增加了对靶向肺部微环境蛋白的吸附，继而能够实现肺部的靶向递送，可以减少药物的使用剂量，有效降低药物治疗带来的毒副作用。
为了使本公开的目的、技术方案和优点更加的清晰明确，以下通过具体实施例结合附图对本公开的技术方案和原理做进一步阐述说明。需要注意的是，下述的具体实施例仅是作为举例说明，本公开的保护范围并不限于此。
下述实施例中所用的试验材料和试剂等，如无特殊说明，均可从商业途径获得。实施例中未注明具体技术或条件者，均为常规方法，可以按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。
实施例中使用的试剂购自国药集团化学试剂有限公司及上海碧云天生物技术有限公司，实施例中使用C57BL/6小鼠购自北京维通利华实验动物技术有限公司。
实施例1
制备永久性阳离子脂质：将5g N-叔丁氧羰基-1，2-乙二胺化合物溶解在无水乙酸乙酯中，随后加入17.25g，125mmol的碳酸钾(K2CO3)和38.125g，125mmol的1-溴十六烷(C16H33Br)。对反应混合物进行回流，用薄层色谱法监测反应过程，48h后，冷却混合物过滤去除碳酸钾残留物。用200ml乙酸乙酯稀释后，用200ml水洗涤3次，加入20ml饱和盐水，使用无水硫酸钠干燥。在减压条件下蒸发得到粗产物，用1％MeOH/CHCl3(v/v)作为洗脱液进行柱层析纯化，得到产物叔丁基2-(二十六烷基氨基)氨基甲酸乙酯。
再将2g，3.28mmol叔丁基2-(二十六烷基氨基)氨基甲酸乙酯溶解于20ml甲基碘中，加入2.0099g，14.542mmol碳酸钾，在室温下搅拌12小时反应得到悬浮液，过滤反应混合物，在旋转蒸发器上蒸发溶剂，随后用乙醚沉淀，得到十六烷基N-甲基十六烷基-1-胺，将2g十六烷基N-甲基十六烷基-1-胺溶解在无水二氯甲烷中，在冰上氮气下滴入5ml三氟乙酸(TFA)，将反应混合物逐渐加热至室温，搅拌4h，确保叔丁基碳基(BOC)完全脱保护。通过重复5次加入50ml氯仿后，在旋转蒸发器上进行浓缩，以完全去除三氟乙酸(TFA)，在 高真空下去除残留溶剂，得到永久性阳离子脂质
图1为本公开实施例1中永久性阳离子脂质和pH响应性脂质的合成示意图。
制备pH响应性脂质：将上述制备过程中得到的叔丁基2-(二十六烷基氨基)氨基甲酸乙酯取2.506g，4.115mmol溶解在无水二氯甲烷中，在冰上氮气下滴入5ml三氟乙酸(TFA)，将反应混合物逐渐加热至室温。搅拌4h，确保叔丁基碳基(BOC)完全脱保护，重复加入50ml氯仿5次，然后在旋转蒸发器上进行浓缩，完全去除溶液中的三氟乙酸(TFA)。在高真空下去除残留溶剂，将产物溶于甲醇，加入等当量的马来酸酐反应得到pH响应性脂质
图2为本公开实施例1中苯硼酸修饰的阳离子脂质的合成示意图。
制备苯硼酸修饰的阳离子脂质：另取烧瓶加入1.0g，16.2mmol 2-氨基乙醇溶于乙酸乙酯，再加入15g，49mmol的1-溴十六烷和2.76g，16.2mmol碳酸钾，在温度85℃下回流48h，回流转速为480rpm。使用普通漏斗过滤碳酸钾，并进行减压蒸发去除溶剂后，将残余物用氯仿萃取，用50ml水洗涤2次分层。将下层有机层用无水硫酸钠干燥2小时，过滤后旋蒸溶剂得到产物2-二十六烷基乙醇。将产物2-二十六烷基乙醇与4-(溴甲基)苯硼酸以摩尔浓度比例1.5:1的比例在N,N-二甲基甲酰胺中混合，并在60℃下搅拌24小时。然后在乙醚中沉淀、干燥得到苯硼酸修饰的阳离子脂质
图3为本公开实施例1中永久性阳离子脂质的电喷雾电离质谱(ESI-MS)图，图4为本公开实施例1中pH响应性脂质的电喷雾电离质谱(ESI-MS)图，图5为本公开实施例1中苯硼酸修饰的阳离子脂质的电喷雾电离质谱(ESI-MS)图。由图3、图4、图5可以看出本公开实施例制备得到的永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的分子 量分别为523.59g/mol、607.57g/mol、644.61g/mol。
实施例2
采用本公开实施例1中制备得到的永久性阳离子脂质pH响应性脂质和苯硼酸修饰的阳离子脂质通过薄膜分散法制备肺靶向纳米胶束，分别按照以下8种不同比例溶解到甲醇溶液，混合在玻璃瓶中。
(1)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为0:3:1(永久性阳离子脂质的摩尔含量为0％)；
(2)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为0.5:2.5:1(永久性阳离子脂质的摩尔含量为12.5％)；
(3)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为1:2:1(永久性阳离子脂质的摩尔含量为25％)；
(4)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为1.5:1.5:1(永久性阳离子脂质的摩尔含量为37.5％)；
(5)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为2:1:1(永久性阳离子脂质的摩尔含量为50％)；
(6)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为2.5:0.5:1(永久性阳离子脂质的摩尔含量为62.5％)；
(7)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为3:0:1(永 久性阳离子脂质的摩尔含量为75％)；
(8)永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为4:0:0(永久性阳离子脂质的摩尔含量为100％)。
使用旋转蒸发仪在100rpm转速和40℃温度减压条件下去除有机溶剂，再玻璃瓶底形成一层均匀薄膜，将玻璃瓶移出并在真空干燥箱中放置2小时以完全去除甲醇；再将1mL超纯水添加到干燥的脂质膜中，进行水合反应得到的脂质悬浮液，对脂质悬浮液进行水浴超声处理约3分钟，形成不同肺靶向纳米胶束。
分别将制备得到的不同肺靶向纳米胶束悬浮液以超纯水进行适当稀释，在25℃下经90Plus PALS高灵敏度Zeta电位及粒度分析仪测定肺靶向纳米胶束的粒径分布和Zeta电位。
图6为本公开实施例2中肺靶向纳米胶束的粒径图，图7为本公开实施例2中肺靶向纳米胶束的Zeta电位图。从图中可以看出本公开实施例制备得到的肺靶向纳米胶束的平均粒径为95.44±17.26nm，随着永久性阳离子脂质的添加比例增加(永久性阳离子脂质的摩尔比从0增加到100％)，肺靶向纳米胶束的Zeta电位由-21.49～+20.91mV变化。
实施例3
将实施例2中以永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质摩尔比例为2:1:1(永久性阳离子脂质的摩尔含量为50％)时，制备得到的肺靶向纳米胶束用移液枪滴1滴于碳膜铜网上，静置1分钟，让样品吸附到碳膜铜网上，用滤纸吸去多余的溶液，用7％乙酸氧铀水溶液染色2分钟，在烘箱中烘干，用透射电子显微镜HT7650在100kV的加速电压下观察观察肺靶向纳米胶束的形貌特征，图8为本公开实施例3中肺靶向纳米胶束的透射电子显微镜(TEM)图。
并将肺靶向纳米胶束在4℃条件下放置7天，图9和图10分别为本公开实施例3中肺靶向纳米胶束粒径稳定性测试图和Zeta电位稳定性测试图，从图中可以看出肺靶向纳米胶束在4℃条件下放置7天其粒径分布及电势未有明显变化，具有较高稳定性。
实施例4
以永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质按摩尔比例为2：1：1进行称重，溶解到甲醇中，再玻璃瓶中混合均匀。取溶解在乙醇浓度为1mM DiD荧光25ul混合到玻璃瓶中，使用旋转蒸发仪在100rpm和40℃减压条件下去除有机溶剂，将玻璃瓶移出并在真空干燥箱中放置2小时以完全去除有机溶剂后，将1mL超纯水添加到干燥的脂质膜中，将得到的脂质悬浮液，对脂质悬浮液进行水浴超声处理约3分钟，在2000rpm条件下低速离心20min去除游离的DiD，取上清液，得到DiD荧光标记的肺靶向纳米胶束。
将DiD荧光标记的肺靶向纳米胶束通过尾静脉注射到C57BL/6健康小鼠体内，在给药后 6小时后断颈处死小鼠，解剖出心、肝、脾、肺、肾等器官。用IVIS Spectrum小动物活体成像仪进行成像检测荧光强度，获得的图片用Living Imaging software进行分析。图11为本公开实施例4中荧光标记的肺靶向纳米胶束的成像分布图，由图11中的体外荧光图像显示可以看出，DiD荧光染料标记的不同阳离子脂质摩尔比的纳米胶束在小鼠体内成像分布，包括解剖小鼠的心、肝、脾、肺、肾等脏器的荧光成像，其中，纳米胶束中的永久性阳离子脂质摩尔比50％时表现出明显的肺靶向性质。
另取DiD荧光标记的肺靶向纳米胶束通过尾静脉注射到小鼠体内，6小时后解剖出器官，然后用IVIS Spectrum小动物活体成像仪进行成像检测荧光强度，确保DiD标记的纳米胶束在小鼠体内。做肺冷冻切片处理，使用激光共聚焦扫描显微镜(LSM880+Airyscan)直接观察肺组织中DiD标记的纳米胶束荧光分布。
图12为本公开实施例4中DiD荧光标记的肺靶向纳米胶束的激光扫描共聚焦显微镜(CLSM)图，图12中DiD表示为DiD荧光标记的肺靶向纳米胶束，FITC-CD31表示为肺内皮细胞，合并图中箭头所指处可以看到DiD荧光标记的肺靶向纳米胶束在肺实质中。
本公开提供的肺靶向纳米胶束、制备方法及应用，通过调整永久性阳离子脂质、pH响应性脂质和苯硼酸修饰的阳离子脂质三种脂质比例组成进而调控纳米胶束的表面电荷性质，使用小鼠器官荧光成像、激光共聚焦显微镜等技术手段证实肺靶向纳米胶束可以特异性的实现肺部靶向递送，从而为实现肺炎高效治疗提供新的手段，也为实现药物在肺组织的靶向递送提供了潜在的机会，可减少药物使用剂量，有效降低药物治疗对其他组织的毒副作用，表现出巨大的临床应用前景。
以上所述本公开的具体实施方式，并不构成对本公开保护范围的限定。任何根据本公开的技术构思所作出的各种其他相应的改变与变形，均应包含在本公开权利要求的保护范围内。

Claims (12)

  1. 一种肺靶向纳米胶束，包括：
    永久性阳离子脂质；
    pH响应性脂质；以及
    苯硼酸修饰的阳离子脂质；
    其中，所述永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质的摩尔比例为X：3-X：1，其中X＝1～2，使得所述纳米胶束被调控为靶向肺部。
  2. 根据权利要求1所述的纳米胶束，其中，
    所述永久性阳离子脂质具有式Ⅰ所示结构：
    其中，R1为C12～C18的烷基。
  3. 根据权利要求1所述的纳米胶束，其中，
    所述pH响应性脂质具有式II所示结构：
    其中，R2为C12～C18的烷基。
  4. 根据权利要求1所述的纳米胶束，其中，
    所述苯硼酸修饰的阳离子脂质具有式III所示结构：
    其中，R3为C12～C18的烷基。
  5. 根据权利要求1所述的纳米胶束，其中，
    所述肺靶向纳米胶束的平均粒径为95.44±17.26nm。
  6. 根据权利要求1所述的纳米胶束，其中，
    所述肺靶向纳米胶束的Zeta电位为-21.49～+20.91mV。
  7. 一种制备如权利要求1～6中任意一项所述的肺靶向纳米胶束的方法，包括：
    将永久性阳离子脂质、pH响应性脂质、苯硼酸修饰的阳离子脂质混合溶解到有机溶剂中，得到混合液；
    除去所述混合液中的所述有机溶剂以形成脂质薄膜，并对所述脂质薄膜进行干燥，得到干燥后的脂质薄膜；
    用超纯水水合处理所述干燥后的脂质薄膜，得到脂质悬浮液后，对所述脂质悬浮液进行水浴超声处理，形成所述肺靶向纳米胶束。
  8. 根据权利要求7所述的方法，其中，
    所述有机溶剂包括甲醇、三氯甲烷中的至少一种。
  9. 根据权利要求7所述的方法，其中，
    除去所述有机溶剂的方法为减压旋蒸，所述减压旋蒸的转速为80～150rpm，所述减压旋蒸的温度为30～60℃；
    所述真空干燥的温度为20～30℃，所述真空干燥的时间为1.5～5h；
    所述超声处理的频率为30～60kHZ，所述超声处理的时间为1～10min。
  10. 一种药物组合物，包含权利要求1～9任一项所述的肺靶向纳米胶束，作为药物载体。
  11. 一种如权利要求1～9任一项所述的肺靶向纳米胶束在制备用于治疗肺部疾病的药物中的应用。
  12. 根据权利要求11所述的应用，其中，
    所述肺部疾病为肺炎。
PCT/CN2023/098249 2023-06-05 2023-06-05 肺靶向纳米胶束、制备方法及应用 Ceased WO2024250133A1 (zh)

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