CN103622906B - High drug-loading capacity amphotericin B polymer composite micelle and preparation method thereof - Google Patents

The invention belongs to the field of biotechnology and particularly relates to a high drug-loading capacity amphotericin B polymer composite micelle and a preparation method thereof. According to the invention, polyethylene glycol-phospholipid with clinical application potentials and indissolvable amphotericin B are used to form amphotericin B polyethylene glycol-phospholipid composite micelle; amphotericin B can combine with polyethylene glycol-phospholipid to form the amphotericin B polymer composite micelle with high drug-loading capacity, high encapsulation efficiency and high stability through the electrostatic attraction function and the interaction between carbon chains. The amphotericin B polymer composite micelle is stable in normal saline and plasma and lower in toxicity and infusion reaction, can be diluted by adopting normal saline, and greatly improves the clinical application range and convenience.

一种高载药量两性霉素B聚合物复合胶束及其制备方法A kind of compound micelle of amphotericin B polymer with high drug load and its preparation method

技术领域technical field

本发明属于医药技术领域，涉及一种高载药量两性霉素B聚合物复合胶束及其制备方法，具体涉及可静脉注射的两性霉素B的纳米复合胶束制剂及其制备方法。The invention belongs to the technical field of medicine, and relates to a high drug-loading amphotericin B polymer composite micelle and a preparation method thereof, in particular to an intravenously injectable nanometer composite micelle preparation of amphotericin B and a preparation method thereof.

背景技术Background technique

两性霉素B属多烯类广谱抗真菌抗生素，主要用于治疗深部真菌感染，并有很高的疗效，在临床已使用了近半个世纪，被称为抗真菌感染的“金标准”。Amphotericin B is a polyene broad-spectrum antifungal antibiotic. It is mainly used to treat deep fungal infections and has a high curative effect. It has been used clinically for nearly half a century and is known as the "gold standard" for antifungal infections. .

两性霉素B抗真菌的作用机制是与真菌细胞膜上的麦角固醇结合，在细胞膜上形成微孔，使细胞内物质外渗而导致真菌死亡。由于两性霉素B具有疏水多烯烃结构和亲水多羟基结构，其在水中的临界聚集浓度非常低（<1.0μg/ml）极容易聚集，并分别以不溶性聚集体、可溶性聚集体和单体三种形式存在。其中，只有以单体形式存在的两性霉素B才能选择性的作用于真菌细胞膜上的麦角甾醇，而聚集体的特异性较低，亦能与哺乳动物细胞膜上的胆固醇结合，引起同样的损伤，故其毒性较大。The antifungal mechanism of amphotericin B is to combine with ergosterol on the fungal cell membrane, form micropores on the cell membrane, and make the intracellular substances extravasate, resulting in the death of the fungus. Because amphotericin B has a hydrophobic polyene structure and a hydrophilic polyhydroxyl structure, its critical aggregation concentration in water is very low (<1.0 μg/ml) and it is easy to aggregate, and it forms insoluble aggregates, soluble aggregates, and monomers, respectively. Three forms exist. Among them, only amphotericin B in the form of monomer can selectively act on ergosterol on the fungal cell membrane, while the specificity of the aggregate is low, and it can also bind to cholesterol on the mammalian cell membrane, causing the same damage , so it is highly toxic.

由于两性霉素B水溶解性质较差，临床上使用脱氧胆酸钠对其进行增溶，作为临床应用的普通注射剂。两性霉素B普通注射剂给药时，药物主要经过肾脏排泄，因此肾毒性较大。此外，两性霉素B普通注射剂在生理盐水中不稳定，会促使红细胞中钾离子渗出，引发各种输液反应例如寒战、发冷、恶心和呕吐。传统两性霉素B注射剂在临床应用时表现出严重的毒副作用，极大地限制了它的应用。因此，开发一种新制剂以达到降低两性霉素B的肾脏毒性和输液反应具有重要的临床意义。Due to the poor solubility of amphotericin B in water, sodium deoxycholate is used clinically to solubilize it as a common injection for clinical application. When amphotericin B ordinary injection is administered, the drug is mainly excreted through the kidneys, so the nephrotoxicity is relatively high. In addition, amphotericin B ordinary injection is unstable in normal saline, which will promote the leakage of potassium ions in red blood cells, causing various infusion reactions such as chills, chills, nausea and vomiting. The traditional amphotericin B injection shows serious side effects in clinical application, which greatly limits its application. Therefore, it is of great clinical significance to develop a new preparation to reduce the renal toxicity and infusion reaction of amphotericin B.

为了降低两性霉素B的毒副作用，国内外研究者一直致力于两性霉素B新制剂的开发，其中最成功的的制剂是两性霉素B脂质体。两性霉素B脂质体于1991年首先在英国和爱尔兰上市，也是第一个脂质体剂型药物。目前，国内上市的脂质体主要有“安浮特克”和“锋克松”。两性霉素B被脂质体包封后，主要分布在肝、脾等网状内皮细胞较丰富的器官中，而在心脏及肾脏中的分布量减小，从而可以减小药物的肾毒性。但两性霉素B脂质体“锋克松”处方中仍含有一定量的表面活性剂去氧胆酸钠，具有一定的溶血毒性，在临床使用过程中存在较大的安全隐患。此外，脂质体制剂存在稳定性差、在储存过程中粒径不稳定、药物包封率低、易泄漏和易形成较大的粒子堵塞毛细血管等缺点。In order to reduce the toxic and side effects of amphotericin B, researchers at home and abroad have been devoting themselves to the development of new formulations of amphotericin B, and the most successful formulation is amphotericin B liposome. Amphotericin B liposome was first launched in the UK and Ireland in 1991, and it was also the first liposomal dosage form drug. At present, the liposomes listed in China mainly include "Amphotec" and "Fengkesong". After amphotericin B is encapsulated by liposomes, it is mainly distributed in organs rich in reticuloendothelial cells such as the liver and spleen, while the distribution in the heart and kidney is reduced, thereby reducing the nephrotoxicity of the drug. However, the prescription of amphotericin B liposome "Fengkesong" still contains a certain amount of surfactant sodium deoxycholate, which has certain hemolytic toxicity, and there is a big safety hazard in the clinical use process. In addition, liposome preparations have disadvantages such as poor stability, unstable particle size during storage, low drug encapsulation efficiency, easy leakage, and easy formation of larger particles to block capillaries.

两亲性嵌段聚合物胶束作为一种新型疏水性药物递送的载体，具有载药范围广、结构稳定、优良的组织渗透性、体内滞留时间长、能有效地携带药物到达靶器官等特点。与脂质体剂型相比，聚合物胶束最明显的优势在于更好的组织渗透性，其原因在于聚合物胶束具有更小的粒径和更柔软的结构。两性霉素B在临床上用于深部组织真菌感染的治疗，渗透性更好的胶束制剂比脂质体制剂具有更好的治疗效果。As a new type of hydrophobic drug delivery carrier, amphiphilic block polymer micelles have the characteristics of wide drug loading range, stable structure, excellent tissue permeability, long residence time in vivo, and can effectively carry drugs to target organs. . Compared with liposomal formulations, the most obvious advantage of polymeric micelles is better tissue penetration, which is due to the smaller particle size and softer structure of polymeric micelles. Amphotericin B is clinically used in the treatment of deep tissue fungal infections, and micellar formulations with better permeability have better therapeutic effects than liposomal formulations.

基于聚乙二醇衍生化磷脂制备的纳米胶束具有的优点包括：1）生物相容性好；2）粒径小，稳定性好，避免了其他微粒给药系统例如脂质体，易于聚集的缺点；3）能够改变药物体内分布，降低药物在肝肾中的蓄积，降低药物的毒副作用；4）延长药物的体内循环时间，增加药物的作用时间和对肿瘤等组织的靶向性。The advantages of nanomicelles prepared based on polyethylene glycol derivatized phospholipids include: 1) good biocompatibility; 2) small particle size and good stability, avoiding other particle drug delivery systems such as liposomes, which are easy to aggregate 3) It can change the distribution of drugs in the body, reduce the accumulation of drugs in the liver and kidney, and reduce the side effects of drugs; 4) Prolong the circulation time of drugs in the body, increase the action time of drugs and the targeting of tumors and other tissues.

发明内容Contents of the invention

本发明目的是针对目前临床现有的两性霉素B制剂如注射用两性霉素B和注射用两性霉素B脂质体的肾毒性、溶血毒性和输液反应等问题，提供一种高载药量两性霉素B聚合物复合胶束给药系统。The purpose of the present invention is to provide a high drug-loading drug for the nephrotoxicity, hemolytic toxicity and transfusion reactions of existing clinical amphotericin B preparations such as amphotericin B for injection and amphotericin B liposome for injection. Quantitative Amphotericin B Polymer Complex Micellar Drug Delivery System.

本发明采用聚乙二醇衍生化磷脂类作为载体材料，通过与一定比例的难溶性药物两性霉素B在水溶液中的相互作用力自组装形成复合骨架胶束，从而显著提高两性霉素B水溶液中的溶解度。其中，载体材料与两性霉素B的摩尔比是0.4～4：1，优选0.5:1～3:1。The present invention uses polyethylene glycol derivatized phospholipids as the carrier material to self-assemble with a certain proportion of insoluble drug amphotericin B in aqueous solution to form composite skeleton micelles, thereby significantly improving the amphotericin B aqueous solution Solubility in . Wherein, the molar ratio of carrier material to amphotericin B is 0.4-4:1, preferably 0.5:1-3:1.

其中所述聚乙二醇衍生化磷脂为聚乙二醇分子通过共价键与磷脂分子上的含氮碱基结合而成。Wherein the polyethylene glycol derivatized phospholipid is formed by combining polyethylene glycol molecules with nitrogen-containing bases on the phospholipid molecules through covalent bonds.

聚乙二醇衍生化磷脂中磷脂部分的脂肪酸包含10～24个碳原子，脂肪酸链是饱和的和部分饱和的，优选月桂酸、肉豆蔻酸、棕榈酸、硬脂酸、油酸或亚油酸。所述的磷脂为磷脂酰乙醇胺、磷脂酰胆碱、磷脂酰肌醇、磷脂酰丝氨酸、二磷脂酰甘油或溶血胆碱磷脂。The fatty acid in the phospholipid part of the polyethylene glycol derivatized phospholipid contains 10 to 24 carbon atoms, and the fatty acid chain is saturated and partially saturated, preferably lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid acid. The phospholipid is phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol or lysocholine phospholipid.

其中聚乙二醇衍生化磷脂的聚乙二醇分子量范围为200～20000，优选500～10000，更优选2000～4000。Wherein the polyethylene glycol molecular weight range of the polyethylene glycol derivatized phospholipid is 200-20000, preferably 500-10000, more preferably 2000-4000.

所述的聚乙二醇衍生化磷脂选自：聚乙二醇2000-磷脂酰乙醇胺、聚乙二醇4000-磷脂酰乙醇胺、聚乙二醇2000-磷脂酰胆碱、聚乙二醇6000-磷脂酰胆碱、聚乙二醇2000-磷脂酰丝氨酸、聚乙二醇4000-磷脂酰丝氨酸、聚乙二醇2000-磷脂酰肌醇、聚乙二醇4000-磷脂酰肌醇、聚乙二醇6000-磷脂酰肌醇，优选：聚乙二醇2000-磷脂酰乙醇胺、聚乙二醇4000-磷脂酰乙醇胺、聚乙二醇2000-磷脂酰肌醇。The polyethylene glycol derivatized phospholipid is selected from: polyethylene glycol 2000-phosphatidylethanolamine, polyethylene glycol 4000-phosphatidylethanolamine, polyethylene glycol 2000-phosphatidylcholine, polyethylene glycol 6000- Phosphatidylcholine, polyethylene glycol 2000-phosphatidylserine, polyethylene glycol 4000-phosphatidylserine, polyethylene glycol 2000-phosphatidylinositol, polyethylene glycol 4000-phosphatidylinositol, polyethylene glycol Alcohol 6000-phosphatidylinositol, preferably: polyethylene glycol 2000-phosphatidylethanolamine, polyethylene glycol 4000-phosphatidylethanolamine, polyethylene glycol 2000-phosphatidylethanolamine.

聚乙二醇衍生化磷脂具有安全、无毒、生物相容性好等优点，并具有较低的临界胶束浓度（10^-6～10^-5mol·L^-1），其形成的胶束经体内血液稀释后仍能保持稳定。因此可以安全地应用于两性霉素B制剂的制备中。Polyethylene glycol derivatized phospholipids have the advantages of safety, non-toxicity, good biocompatibility, etc., and have a low critical micelle concentration (10 ^-6 ~ 10 ^-5 mol·L ^-1 ), the micelles formed by it It remains stable after blood dilution in vivo. Therefore, it can be safely used in the preparation of amphotericin B preparations.

本发明的聚乙二醇衍生化磷脂与药物两性霉素B的复合胶束，可通过以下方法制备：薄膜分散法和冻干法。The composite micelle of polyethylene glycol derivatized phospholipid and drug amphotericin B of the present invention can be prepared by the following methods: film dispersion method and freeze-drying method.

薄膜分散法包括以下步骤：The thin film dispersion method includes the following steps:

（1）将两性霉素B和聚乙二醇衍生化磷脂的混合物溶于有机溶剂中；(1) dissolving the mixture of amphotericin B and polyethylene glycol derivatized phospholipids in an organic solvent;

（2）将以上混合物在真空中旋转挥干，得聚乙二醇衍生化磷脂的与两性霉素B的混合物薄膜；(2) Rotate and evaporate the above mixture to dryness in a vacuum to obtain a mixture film of polyethylene glycol derivatized phospholipid and amphotericin B;

（3）加入适量去离子水，复溶即得均匀澄清的胶束溶液。(3) Add an appropriate amount of deionized water and redissolve to obtain a uniform and clear micellar solution.

冻干法包括以下步骤：Freeze-drying involves the following steps:

（1）将两性霉素B和聚乙二醇衍生化磷脂的混合物溶于有机溶剂中；(1) dissolving the mixture of amphotericin B and polyethylene glycol derivatized phospholipids in an organic solvent;

（2）加入适量冻干保护剂，经过冷冻干燥去除水分，得干燥的包载疏水抗肿瘤药物两性霉素B的聚合物胶束；(2) adding an appropriate amount of freeze-drying protective agent, and removing water by freeze-drying to obtain dry polymer micelles loaded with hydrophobic anti-tumor drug amphotericin B;

（3）将制得的冻干胶束按需要量加水，水合振荡后，复原成胶束溶液。(3) Add water to the prepared freeze-dried micelles according to the required amount, and after hydration and shaking, restore to a micellar solution.

所述去离子水包括：蒸馏水、生理盐水、等渗葡萄糖溶液或缓冲溶液The deionized water includes: distilled water, physiological saline, isotonic glucose solution or buffer solution

所述的有机溶剂为甲醇、乙醇、氯仿、丙酮、二甲基亚砜一种或其混合溶剂。The organic solvent is one of methanol, ethanol, chloroform, acetone, dimethyl sulfoxide or a mixed solvent thereof.

冻干保护剂包括葡萄糖、甘露醇、蔗糖、海藻糖、麦芽糖、聚乙二醇2000、聚乙二醇4000、聚乙二醇6000或普朗尼克F68。Lyoprotectants include glucose, mannitol, sucrose, trehalose, maltose, polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000 or Pluronic F68.

通过上述方法制备的聚合物胶束组合物可通过高压乳匀、高速均质或超声等工艺减小胶束的粒径和粒径分布。该聚合物胶束组合物亦可经一定制剂工艺，如冷冻干燥、喷雾干燥、减压蒸发等进一步制备成适宜制剂，如注射剂、口服制剂等。The polymer micelle composition prepared by the above method can reduce the particle size and particle size distribution of the micelles by high-pressure emulsification, high-speed homogenization or ultrasonic technology. The polymer micelle composition can also be further prepared into suitable preparations, such as injections and oral preparations, through certain preparation processes, such as freeze-drying, spray-drying, and evaporation under reduced pressure.

本发明通过红外光谱法鉴定了载体聚乙二醇衍生化磷脂与药物两性霉素B存在相互作用。使用红外光谱仪分别对载药胶束、聚乙二醇衍生化磷脂、两性霉素B及其混合物进行扫描，通过考察化合物特征基团的吸收峰峰位的变化，确证了化合物周围电子环境的变化，确定了两者之间存在相互作用。推测聚乙二醇-磷脂酰乙醇胺羰基与两性霉素B的羟基存在氢键物理相互作用。The present invention identifies the interaction between the carrier polyethylene glycol derivatized phospholipid and the drug amphotericin B by means of infrared spectroscopy. Use infrared spectrometer to scan the drug-loaded micelles, polyethylene glycol derivatized phospholipids, amphotericin B and their mixtures respectively, and confirm the changes of the electronic environment around the compounds by examining the changes in the absorption peak positions of the characteristic groups of the compounds , confirming that there is an interaction between the two. It is speculated that there is a hydrogen bond physical interaction between the polyethylene glycol-phosphatidylethanolamine carbonyl and the hydroxyl group of amphotericin B.

本发明通过测定粒径，考察了载药胶束在生理盐水中的稳定性。临床上使用的注射用两性霉素B和注射用两性霉素B脂质体都只能使用葡萄糖溶液进行稀释，经生理盐水稀释会产生沉淀。而本发明所制备的产品可在大量生理盐水内，保持长时间的稳定性，提高了临床使用的范围及方便性。In the present invention, the stability of the drug-loaded micelles in physiological saline is investigated by measuring the particle size. Clinically used amphotericin B for injection and amphotericin B liposome for injection can only be diluted with glucose solution, and precipitation will occur when diluted with normal saline. However, the product prepared by the present invention can maintain long-term stability in a large amount of physiological saline, which improves the scope and convenience of clinical use.

本发明通过包封率、载药量和粒径考察了载药胶束冻干粉的稳定性。复合胶束在冻干保护剂的保护作用下，稳定性良好，药物不易泄露，粒子不易聚集。The present invention investigates the stability of the drug-loaded micelles freeze-dried powder through encapsulation efficiency, drug-loading capacity and particle size. Under the protection of the freeze-drying protective agent, the composite micelles have good stability, the drugs are not easy to leak, and the particles are not easy to aggregate.

本发明考察了两性霉素B复合胶束的体外释放情况。两性霉素B复合胶束具有明显的缓释特点，释药平稳无突释效应。胶束载体可有效包裹两性霉素B，避免制剂在血液循环过程中药物快速释放，从而降低药物的毒副作用；可以显著延长药物作用时间，达到长效作用。The present invention investigates the in vitro release of amphotericin B complex micelles. Amphotericin B complex micelles have obvious sustained-release characteristics, and the drug release is stable without burst release effect. The micellar carrier can effectively encapsulate the amphotericin B, avoiding the rapid release of the drug during the blood circulation of the preparation, thereby reducing the toxic and side effects of the drug; it can significantly prolong the action time of the drug and achieve a long-acting effect.

本发明通过对溶血率的测定考察了载药胶束的体外溶血毒性，与两性霉素B的二甲基亚砜溶液、注射用两性霉素B和注射用两性霉素B脂质体溶液相比，两性霉素B聚合物复合胶束溶血毒性显著降低。由于两性霉素B复合胶束能够缓慢释放药物，避免药物聚集而产生溶血毒性；复合胶束可以使两性霉素B以毒性较低的单体形式释放，降低溶血毒性。The present invention investigates the in vitro hemolytic toxicity of the drug-loaded micelles by measuring the hemolysis rate, which is compatible with the dimethyl sulfoxide solution of amphotericin B, amphotericin B for injection and amphotericin B liposome solution for injection Compared with that, the hemolytic toxicity of amphotericin B polymer complex micelles was significantly reduced. Since the amphotericin B complex micelle can slowly release the drug, avoiding the hemolytic toxicity caused by drug aggregation; the compound micelle can release the amphotericin B in the form of a less toxic monomer, reducing the hemolytic toxicity.

本发明通过液基稀释法测定了载药胶束的MIC₈₀值，考察了体外抗菌活性。其体外抗菌活性优于两性霉素B的二甲基亚砜溶液和注射用两性霉素B溶液。两性霉素B的二甲基亚砜溶液和注射用两性霉素B溶液在进行稀释时，部分两性霉素B可能形成聚集体，导致其有效浓度降低；而复合胶束的耐稀释性较好，药物释放缓慢，不易聚集，因而有效浓度较高。此外，复合胶束可在一定程度上防止两性霉素B的多烯烃基团氧化，保持药物的活性。In the present invention, the MIC ₈₀ value of the drug-loaded micelles is measured by a liquid-based dilution method, and the antibacterial activity in vitro is investigated. Its in vitro antibacterial activity is superior to amphotericin B dimethyl sulfoxide solution and amphotericin B solution for injection. When the dimethyl sulfoxide solution of amphotericin B and the amphotericin B solution for injection are diluted, some amphotericin B may form aggregates, resulting in a decrease in its effective concentration; while the compound micelles have better dilution resistance , The drug releases slowly and is not easy to aggregate, so the effective concentration is higher. In addition, the complex micelles can prevent the oxidation of the polyene groups of amphotericin B to a certain extent, maintaining the activity of the drug.

附图说明Description of drawings

图1为聚乙二醇衍生化磷脂酰乙醇胺的¹H-NMR图谱Figure 1 is the ¹ H-NMR spectrum of polyethylene glycol derivatized phosphatidylethanolamine

图2为两性霉素B的¹H-NMR图谱Figure 2 is the ¹ H-NMR spectrum of amphotericin B

图3为两性霉素B与聚乙二醇衍生化磷脂酰乙醇胺混合物的¹H-NMR图谱Figure 3 is the ¹ H-NMR spectrum of the mixture of amphotericin B and polyethylene glycol derivatized phosphatidylethanolamine

图4为实施例3所制备的复合胶束的¹H-NMR图谱Figure 4 is the ¹ H-NMR spectrum of the composite micelles prepared in Example 3

图5为聚乙二醇2000-磷脂酰乙醇胺的IR图谱Figure 5 is the IR spectrum of polyethylene glycol 2000-phosphatidylethanolamine

图6为两性霉素B的IR图谱Figure 6 is the IR spectrum of amphotericin B

图7为两性霉素B与聚乙二醇2000-磷脂酰乙醇胺混合物的IR图谱Figure 7 is the IR spectrum of amphotericin B and polyethylene glycol 2000-phosphatidylethanolamine mixture

图8为实施例3所制备的复合胶束的IR图谱Fig. 8 is the IR spectrum of the composite micelle prepared in embodiment 3

图9为实施例1～6产品72h内的释放曲线Fig. 9 is the release curve in 72h of embodiment 1～6 product

图10两性霉素B的二甲基亚砜溶液、注射用两性霉素B溶液、注射用两性霉素B脂质体溶液和实施例3溶液的溶血情况The hemolysis situation of the dimethyl sulfoxide solution of Fig. 10 amphotericin B, amphotericin B solution for injection, amphotericin B liposome solution for injection and embodiment 3 solution

具体实施方式detailed description

根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的发明。The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the invention described in the claims.

实施例1：聚乙二醇2000-磷脂酰乙醇胺与两性霉素B的复合胶束制备（摩尔比为0.4:1）Example 1: Preparation of composite micelles of polyethylene glycol 2000-phosphatidylethanolamine and amphotericin B (molar ratio is 0.4:1)

称取8.4mg两性霉素B和10mg聚乙二醇2000-磷脂酰乙醇胺溶于20ml甲醇，在50℃水浴中减压旋转蒸发除去有机溶剂，加入蒸馏水10ml，水化15分钟，过0.22μm微孔滤膜过滤，即得复合胶束。测得其包封率为95.8%，载药量为44.4%，粒径为57.89nm。Weigh 8.4 mg of amphotericin B and 10 mg of polyethylene glycol 2000-phosphatidylethanolamine and dissolve it in 20 ml of methanol, remove the organic solvent by rotary evaporation under reduced pressure in a water bath at 50 ° C, add 10 ml of distilled water, hydrate for 15 minutes, and pass through a micrometer of 0.22 μm Pore membrane filtration, that is, composite micelles. It was measured that the encapsulation efficiency was 95.8%, the drug loading was 44.4%, and the particle size was 57.89nm.

实施例2：聚乙二醇2000-磷脂酰乙醇胺和两性霉素B的复合胶束制备（摩尔比为4:1）Example 2: Preparation of composite micelles of polyethylene glycol 2000-phosphatidylethanolamine and amphotericin B (molar ratio is 4:1)

称取0.8mg两性霉素B和10mg聚乙二醇2000-磷脂酰乙醇胺溶于10ml甲醇-二甲基亚砜（1:1），在50℃水浴中减压旋转蒸发除去有机溶剂，加入磷酸缓冲盐10ml水化20分钟，过0.22μm微孔滤膜过滤，加入20mg甘露醇作为冻干保护剂，冷冻干燥。用注射用水复溶，测得其包封率为99.5%，载药量为7.4%，粒径为60.03nm。Weigh 0.8mg of amphotericin B and 10mg of polyethylene glycol 2000-phosphatidylethanolamine and dissolve in 10ml of methanol-dimethylsulfoxide (1:1), remove the organic solvent by rotary evaporation under reduced pressure in a water bath at 50°C, and add phosphoric acid 10 ml of buffer salt was hydrated for 20 minutes, filtered through a 0.22 μm microporous membrane, 20 mg of mannitol was added as a freeze-drying protective agent, and freeze-dried. Reconstituted with water for injection, the encapsulation rate was 99.5%, the drug loading was 7.4%, and the particle size was 60.03nm.

实施例3：聚乙二醇4000-磷脂酰乙醇胺和两性霉素B的复合胶束制备（摩尔比为0.5:1）Example 3: Preparation of composite micelles of polyethylene glycol 4000-phosphatidylethanolamine and amphotericin B (molar ratio is 0.5:1)

称取6.6mg两性霉素B和10mg聚乙二醇4000-磷脂酰乙醇胺溶于18ml甲醇，在60℃水浴中减压旋转蒸发除去有机溶剂，加入蒸馏水10ml水化15分钟，过0.22μm微孔滤膜过滤，即得复合胶束。测得其包封率为98.7%，载药量为39.8%，粒径为62.02nm。Weigh 6.6mg of amphotericin B and 10mg of polyethylene glycol 4000-phosphatidylethanolamine and dissolve it in 18ml of methanol, remove the organic solvent by rotary evaporation under reduced pressure in a 60°C water bath, add 10ml of distilled water to hydrate for 15 minutes, and pass through a 0.22μm micropore Membrane filtration to obtain complex micelles. It was measured that the encapsulation efficiency was 98.7%, the drug loading capacity was 39.8%, and the particle size was 62.02nm.

实施例4：聚乙二醇2000-磷脂酰肌醇和两性霉素B复合胶束的制备（摩尔比为3:1）Example 4: Preparation of polyethylene glycol 2000-phosphatidylinositol and amphotericin B complex micelles (molar ratio is 3:1)

称取1.1mg两性霉素B和10mg聚乙二醇2000-磷脂酰肌醇胺溶于18ml甲醇，在60℃水浴中减压旋转蒸发除去有机溶剂，加入蒸馏水10ml水化15分钟，过0.22μm微孔滤膜过滤，即得复合胶束。测得其包封率为99.8%，载药量为9.9%，粒径为63.42nm。Weigh 1.1mg of amphotericin B and 10mg of polyethylene glycol 2000-phosphatidylinositolamine and dissolve it in 18ml of methanol, remove the organic solvent by rotary evaporation under reduced pressure in a water bath at 60°C, add 10ml of distilled water for hydration for 15 minutes, and pass through 0.22μm Filter through a microporous membrane to obtain complex micelles. It was measured that the encapsulation efficiency was 99.8%, the drug loading capacity was 9.9%, and the particle size was 63.42nm.

实施例5：聚乙二醇4000-磷脂酰乙醇胺和两性霉素B的复合胶束制备（摩尔比为1:1）Example 5: Preparation of composite micelles of polyethylene glycol 4000-phosphatidylethanolamine and amphotericin B (molar ratio is 1:1)

称取3.3mg两性霉素B和10mg聚乙二醇2000-磷脂酰乙醇胺溶于10ml甲醇-二甲基亚砜（1:1），在50℃水浴中减压旋转蒸发除去有机溶剂，加入磷酸缓冲盐10ml水化20分钟，过0.22μm微孔滤膜过滤，加入20mg甘露醇作为冻干保护剂，冷冻干燥。用注射用水复溶，测得其包封率为99.5%，载药量为24.8%，粒径为58.62nm。Weigh 3.3mg of amphotericin B and 10mg of polyethylene glycol 2000-phosphatidylethanolamine and dissolve in 10ml of methanol-dimethyl sulfoxide (1:1), remove the organic solvent by rotary evaporation under reduced pressure in a water bath at 50°C, and add phosphoric acid 10 ml of buffer salt was hydrated for 20 minutes, filtered through a 0.22 μm microporous membrane, 20 mg of mannitol was added as a freeze-drying protective agent, and freeze-dried. Reconstituted with water for injection, the encapsulation rate was 99.5%, the drug loading was 24.8%, and the particle size was 58.62nm.

实施例6：聚乙二醇4000-磷脂酰乙醇胺和两性霉素B的复合胶束制备（摩尔比为2:1）Example 6: Preparation of composite micelles of polyethylene glycol 4000-phosphatidylethanolamine and amphotericin B (molar ratio is 2:1)

称取1.7mg两性霉素B和10mg聚乙二醇2000-磷脂酰乙醇胺溶于10ml甲醇，在50℃水浴中减压旋转蒸发除去有机溶剂，加入蒸馏水10ml，水化15分钟，过0.22μm微孔滤膜过滤，即得复合胶束。测得其包封率为99.8%，载药量为14.5%，粒径为56.89nm。Weigh 1.7mg of amphotericin B and 10mg of polyethylene glycol 2000-phosphatidylethanolamine and dissolve it in 10ml of methanol, remove the organic solvent by rotary evaporation under reduced pressure in a water bath at 50°C, add 10ml of distilled water, hydrate for 15 minutes, pass through a 0.22μm micrometer Pore membrane filtration, that is, composite micelles. It was measured that the encapsulation efficiency was 99.8%, the drug loading was 14.5%, and the particle size was 56.89nm.

实施例7：按实施例3法制备的载药胶束，冷冻干燥后，称取1.0mg溶于0.5ml氘水中，使用Mercury Plus400MHz核磁共振波谱仪鉴定，获得核磁共振图谱（如图4）。分别称取1.0mg的聚乙二醇衍生化磷脂酰乙醇胺、两性霉素B和两者的混合物（mol:mol1:2）溶于0.5ml氘代二甲基亚砜中，使用Mercury Plus400MHz核磁共振波谱仪鉴定，获得核磁共振图谱（如图1、2和3）。如图可知，形成载药胶束后，聚合物磷脂端的核磁振动吸收峰消失，只显示了聚乙二醇端的吸收峰，表明聚乙二醇衍生化磷脂酰乙醇胺形成了稳定的核壳结构；两性霉素B的核磁振动吸收峰消失，表明两性霉素B被包载在胶束核内。Example 7: After freeze-drying the drug-loaded micelles prepared by the method in Example 3, weigh 1.0 mg and dissolve it in 0.5 ml of deuterium water, use Mercury Plus 400 MHz nuclear magnetic resonance spectrometer to identify, and obtain nuclear magnetic resonance spectrum (as shown in Figure 4). Weigh 1.0mg of polyethylene glycol derivatized phosphatidylethanolamine, amphotericin B and the mixture of the two (mol:mol1:2) and dissolve them in 0.5ml deuterated dimethyl sulfoxide, and use Mercury Plus400MHz nuclear magnetic resonance The spectrometer was identified and the nuclear magnetic resonance spectrum was obtained (as shown in Figures 1, 2 and 3). As can be seen from the figure, after the formation of drug-loaded micelles, the NMR vibration absorption peak of the polymer phospholipid end disappeared, and only the absorption peak of the polyethylene glycol end was shown, indicating that the polyethylene glycol derivatized phosphatidylethanolamine formed a stable core-shell structure; The NMR vibration absorption peak of amphotericin B disappeared, indicating that amphotericin B was entrapped in the micelle core.

实施例8：按实施例3法制备的载药胶束，冷冻干燥后，采用Brukervector22型红外光谱仪分别测定聚乙二醇2000-磷脂酰乙醇胺、两性霉素B、实施例3制备的冻干粉的红外光谱，方法是固体样品和溴化钾粉末混合研磨，进行压片，然后直接进行测试。扫描所得红外图谱如5～8，由图可知聚乙二醇-磷脂酰乙醇胺位于1738cm^-1处(-C=O)的吸收峰位移至1730cm^-1，两性霉素B位于3411cm处(-OH)的吸收峰位移至3419cm^-1，表明两者之间具有相互作用。Embodiment 8: The drug-loaded micelles prepared by the method of Example 3, after freeze-drying, adopt the Brukervector22 type infrared spectrometer to measure polyethylene glycol 2000-phosphatidylethanolamine, amphotericin B, and the freeze-dried powder prepared in Example 3 respectively Infrared spectroscopy, the method is that the solid sample and potassium bromide powder are mixed and ground, pressed into tablets, and then tested directly. The infrared spectrum obtained by scanning is shown in 5-8. It can be seen from the figure that the absorption peak of polyethylene glycol-phosphatidylethanolamine at 1738cm ^-1 (-C=O) shifts to 1730cm ^-1 , and that of amphotericin B at 3411cm (-OH ) shifted to 3419cm ^-1 , indicating that there is an interaction between the two.

实施例9：复合胶束包封率和载药量的测定方法Embodiment 9: Determination method of compound micelle encapsulation efficiency and drug loading

精密吸取0.5ml实施例1～6的产品溶液，缓缓加于葡聚糖凝胶柱上，以3000r/min离心2分钟，收集前三管滤液，合并，甲醇稀释，采用紫外分光光度计测定，按下式计算包封率和载药量：Precisely draw 0.5ml of the product solution of Examples 1 to 6, slowly add it to the Sephadex column, centrifuge at 3000r/min for 2 minutes, collect the first three tubes of filtrate, combine, dilute with methanol, and measure with a UV spectrophotometer , calculate the encapsulation efficiency and drug loading according to the formula:

其中，W₁为包裹在胶束中的药物质量；W₂为投药量，W₃为载体材料质量。测得实施例1～6所制备的产品的包封率和载药量如表1。表中数据表明。Among them, W ₁ is the mass of the drug encapsulated in the micelles; W ₂ is the dosage, and W ₃ is the mass of the carrier material. The measured encapsulation efficiency and drug loading capacity of the products prepared in Examples 1-6 are shown in Table 1. The data in the table shows.

表1实例1～6产品的包封率和载药量Encapsulation efficiency and drug loading of table 1 example 1～6 products

实施例10：两性霉素B复合胶束在生理盐水中的稳定性研究Example 10: Stability study of amphotericin B complex micelles in physiological saline

将实施例1～6所制备的产品分别用100ml生理盐水进行稀释，使用马尔文粒径测定仪分别于0.5、1、2、4、6、12、24小时后测定其粒径。粒径测定结果如表2。The products prepared in Examples 1-6 were respectively diluted with 100 ml of normal saline, and their particle sizes were measured after 0.5, 1, 2, 4, 6, 12, and 24 hours using a Malvern particle size analyzer. The particle size measurement results are shown in Table 2.

表2.两性霉素B复合胶束在生理盐水中的稳定性Table 2. Stability of amphotericin B complex micelles in saline

实施例11：两性霉素B复合胶束冻干粉末稳定性的研究Example 11: Study on Stability of Amphotericin B Complex Micelle Freeze-dried Powder

将实施例3所制备的两性霉素B复合胶束，加入冻干保护剂，冷冻干燥后于室温放置，分别于1、2、4、6、8、10、20、30天用注射用水复溶，并测定其包封率、载药量和粒径。测定结果如表3。The amphotericin B complex micelles prepared in Example 3 were added with a lyoprotectant, placed at room temperature after freeze-drying, and reconstituted with water for injection on 1, 2, 4, 6, 8, 10, 20, and 30 days respectively. dissolved, and its encapsulation efficiency, drug loading and particle size were determined. The measurement results are shown in Table 3.

表3.两性霉素B复合胶束冻干粉的稳定性Table 3. Stability of amphotericin B complex micellar lyophilized powder

实施例12：两性霉素B胶束体外释放的考察Example 12: Investigation of in vitro release of amphotericin B micelles

取实施例1～6所制备的两性霉素B胶束各2ml，置于透析袋（分子量12000～15000）内，再放入装有200mL pH10.0PBS释放介质的锥形瓶内。将锥形瓶置于恒温振荡器内，于37℃、100r/min的条件上进行释药实验。分别于0.5、1、2、4、6、12、24、36、48、72h取透析外液，同时补加等量的相同温度的释放介质。使用紫外分光光度计测定药物释放量，绘制释放曲线如图9。Take 2ml each of the amphotericin B micelles prepared in Examples 1-6, place them in a dialysis bag (molecular weight 12000-15000), and then put them into a conical flask filled with 200mL of pH10.0 PBS release medium. The Erlenmeyer flask was placed in a constant temperature shaker, and the drug release experiment was carried out under the conditions of 37°C and 100r/min. At 0.5, 1, 2, 4, 6, 12, 24, 36, 48, and 72 hours, the dialyzed fluid was taken, and an equal amount of release medium at the same temperature was added at the same time. Use the ultraviolet spectrophotometer to measure the drug release amount, and draw the release curve as shown in Figure 9.

结果表明，两性霉素B复合胶束具有非常明显的缓释特征。The results showed that the amphotericin B complex micelles had very obvious slow-release characteristics.

实施例13：两性霉素B复合胶束体外溶血性的考察Example 13: In vitro hemolytic investigation of amphotericin B complex micelles

取健康新西兰大耳兔1只，耳缘静脉取血，置于经肝素处理的试管内，加入适量0.9%氯化钠注射液洗涤。2000r/min离心10min,弃去上清液。重复上述操作数次,直至离心后上清液呈无色透明。弃去上清液，将所得红细胞加入0.9%氯化钠注射液进行稀释，配制成2%的红细胞混悬液，备用。One healthy New Zealand big-eared rabbit was taken, and blood was collected from the ear vein, placed in a heparin-treated test tube, and washed with an appropriate amount of 0.9% sodium chloride injection. Centrifuge at 2000r/min for 10min, discard the supernatant. Repeat the above operation several times until the supernatant is colorless and transparent after centrifugation. The supernatant was discarded, and the obtained erythrocytes were diluted with 0.9% sodium chloride injection to prepare a 2% erythrocyte suspension for later use.

将实施例3所制备的载药胶束、注射用两性霉素B、注射用两性霉素B脂质体和两性霉素B的游离溶液使用生理盐水稀释，配制浓度范围为2～256μg/ml的系列浓度溶液。精密吸取上述溶液2.5ml，置于试管中，然后加入2%红细胞混悬液2.5ml，混匀，于37℃下培养3h后，将样品溶液置于冰浴中，终止溶血反应，以2000r/min离心10min,吸取上清液,于540nm处测定其吸光度，按下述公式计算溶血率:The drug-loaded micelles prepared in Example 3, amphotericin B for injection, amphotericin B liposome for injection and the free solution of amphotericin B were diluted with physiological saline, and the prepared concentration range was 2-256 μg/ml series of concentration solutions. Precisely draw 2.5ml of the above solution, put it in a test tube, then add 2.5ml of 2% erythrocyte suspension, mix well, incubate at 37°C for 3 hours, put the sample solution in an ice bath to stop the hemolysis reaction, and set the temperature at 2000r/ Centrifuge for 10 min, draw the supernatant, measure its absorbance at 540 nm, and calculate the hemolysis rate according to the following formula:

四种溶液在3h时的溶血情况如图10。实施例3所制备胶束制剂溶血率显著低于其他三种溶液。实施例14：两性霉素B复合胶束体外抗菌效果的考察The hemolysis of the four solutions at 3 hours is shown in Figure 10. The hemolysis rate of the micellar preparation prepared in Example 3 was significantly lower than that of the other three solutions. Example 14: Investigation of the antibacterial effect of amphotericin B complex micelles in vitro

采用RPMI1640液体培养基稀释白色念珠菌溶液，配制成浓度约0.5～2.5×10³CFU/mL的接种菌液。将实施例3所制备的胶束，注射用两性霉素B和两性霉素B的游离溶液用RPMI1640液体培养基进行倍比稀释。将药物溶液与空白对照溶液分别加入96孔板中，再加入0.1mL含菌培养液。将96孔板置于恒温培养箱中35℃培养48h，观察结果。用酶联免疫监测仪在540nm处测定96孔板各孔的光密度值。计算MIC值如表3。The Candida albicans solution was diluted with RPMI1640 liquid medium to prepare an inoculum solution with a concentration of about 0.5-2.5×10 ³ CFU/mL. The micelles prepared in Example 3, the amphotericin B for injection and the free solution of amphotericin B were diluted with RPMI1640 liquid medium. The drug solution and the blank control solution were added to the 96-well plate, and then 0.1 mL of the culture medium containing bacteria was added. The 96-well plate was placed in a constant temperature incubator at 35°C for 48 hours, and the results were observed. The optical density value of each well of the 96-well plate was measured at 540 nm with an enzyme-linked immunosorbent monitor. The calculated MIC values are shown in Table 3.

表3三种溶液的体外抗真菌结果The antifungal results in vitro of three kinds of solutions of table 3

综上所述，两性霉素B复合胶束与现有普通注射剂和脂质体相比，具有明显的优势，主要体现在如下几个方面：1.载药量高，本发明的两性霉素B聚合物胶束制剂中药物的浓度可达1.2mg/ml，显著高于普通注射液、两性霉素B脂质体；2.本发明的两性霉素B聚合物胶束制剂溶血性明显小于普通注射液和两性霉素B脂质体；3.本发明的两性霉素B聚合物胶束制剂的体外抗真菌效果明显好于普通注射液；4.本发明的两性霉素B聚合物胶束制剂缓释效果明显好于两性霉素B脂质体。In summary, compared with existing common injections and liposomes, amphotericin B composite micelles have obvious advantages, which are mainly reflected in the following aspects: 1. The drug loading is high, and the amphotericin of the present invention The concentration of medicine in the B polymer micelle preparation can reach 1.2mg/ml, significantly higher than common injection, amphotericin B liposome; 2. the amphotericin B polymer micelle preparation of the present invention is obviously less than Ordinary injection and amphotericin B liposome; 3. the in vitro antifungal effect of amphotericin B polymer micelle preparation of the present invention is obviously better than ordinary injection; 4. amphotericin B polymer glue of the present invention The sustained-release effect of bundle preparation is obviously better than that of amphotericin B liposome.