CN112545994B - A kind of simultaneous preparation method of small particle size, high stability multi-chamber and single-chamber liposome - Google Patents

Abstract

Translated fromChinese

本发明涉及一种小粒径、高稳定性多室及单室脂质体的同步制备方法，属于化工和制药领域，本发明以脂质体粒径为控制对象，利用单因素实验对影响粒径的工艺条件进行了优化，采用改良薄膜分散法制备了粒径约110 nm的多室脂质体，并通过上调水合温度55℃，实现了多室脂质体向单室脂质体的转变，并利用动态光散射（DLS）和透射电子显微镜（TEM）技术检测了多室和单室脂质体的分散性和形态学特征。该制备工艺简单，可同时实现多室脂质体和单室脂质体的制备，制得的多室和单室脂质体粒径均一、表面光滑、分散性良好，且具有良好的储存稳定性。

The invention relates to a synchronous preparation method of multi-chamber and single-chamber liposomes with small particle size and high stability, belonging to the fields of chemical industry and pharmacy. The process conditions of the diameter were optimized, and the multilamellar liposomes with a particle size of about 110 nm were prepared by the modified film dispersion method. , and examined the dispersion and morphological characteristics of multi- and unilamellar liposomes using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The preparation process is simple, the preparation of multi-chamber liposome and unilamellar liposome can be realized at the same time, the prepared multi-chamber liposome and unilamellar liposome have uniform particle size, smooth surface, good dispersibility and good storage stability sex.

Description

Translated fromChinese

一种小粒径、高稳定性多室及单室脂质体的同步制备方法A kind of simultaneous preparation method of small particle size, high stability multi-chamber and single-chamber liposome

技术领域technical field

本发明属于化工与制药技术领域，具体地，涉及一种多室及单室脂质体的同步制备方法。The invention belongs to the technical field of chemical industry and pharmacy, and in particular relates to a simultaneous preparation method of multi-chamber and single-chamber liposomes.

背景技术Background technique

脂质体（liposome）是由大豆卵磷脂和胆固醇等两亲性分子组成的双分子层脂质囊泡，其粒径从10 nm到2 μm不等。根据粒径和层数的不同，脂质体可以分为以下3类：粒径10~100 nm的单层脂质体称为小单室脂质体（small unilamellar vesicles，SUVs）；粒径100 nm~1 μm的单层脂质体称为大单室脂质体（large unilamellar vesicles，LUVs）；粒径1~5 μm的多层囊泡称为多室脂质体（multilamellar lipid vesicles，MLVs）。其中，小粒径的多室脂质体比较少见。脂质体的粒径是决定其稳定性和生物体内应用的关键参数。多室脂质体由几个脂质同心圆囊泡叠加而成，由于脂质层数较多，脂质层间隙脂溶性药物的包封率高，但层与层之间距离较小容易发生膜融合从而使脂质体粒径变大且不稳定。粒径过大的单室脂质体存在稳定性差，粒径大小不均等问题。通常，脂质体的粒径越小，脂质体越稳定，粒径100 nm左右的脂质体由于在肿瘤部位具有较好的EPR效应（EnhancedPermeability and Retention Effect）而受到极大青睐。另外，脂质体粒径的大小还直接影响载体是否能够顺利进入组织和细胞发挥作用。Liposomes are bilayer lipid vesicles composed of amphiphilic molecules such as soybean lecithin and cholesterol, with particle sizes ranging from 10 nm to 2 μm. Liposomes can be divided into the following three categories according to their particle size and number of layers: unilamellar liposomes with a particle size of 10–100 nm are called small unilamellar vesicles (SUVs); Unilamellar vesicles with a size of nm~1 μm are called large unilamellar vesicles (LUVs); multilamellar vesicles with a particle size of 1~5 μm are called multilamellar lipid vesicles (MLVs). ). Among them, multilamellar liposomes with small particle size are relatively rare. The particle size of liposomes is a key parameter that determines its stability and in vivo application. Multilamellar liposomes are formed by stacking several lipid concentric vesicles. Due to the large number of lipid layers, the encapsulation efficiency of lipid-soluble drugs between lipid layers is high, but the distance between layers is small and prone to occur. Membrane fusion results in large and unstable liposomes. Unilamellar liposomes with too large particle size have problems such as poor stability and uneven particle size. Generally, the smaller the particle size of liposomes, the more stable the liposomes are. Liposomes with a particle size of about 100 nm are greatly favored due to their better EPR effect (Enhanced Permeability and Retention Effect) at the tumor site. In addition, the size of the liposome particle size also directly affects whether the carrier can smoothly enter tissues and cells to play a role.

影响脂质体粒径的因素包括制备脂质体时所用的磷脂种类、磷脂与胆固醇的比例以及磷脂的相变温度（Tc）等。相变温度是影响脂质体形态的很重要的因素之一：当温度低于磷脂相变温度时，脂质体中磷脂之间的排列较为紧凑，脂质体状态稳定；当环境温度高于磷脂相变温度时，磷脂排列松散，脂质体内部及脂质体之间的流动性增大。除此之外，对脂质体进行机械挤压、离心或过凝胶色谱柱等物理方法也可以较好控制脂质体粒径大小。Factors affecting the particle size of liposomes include the type of phospholipids used in the preparation of liposomes, the ratio of phospholipids to cholesterol, and the phase transition temperature (Tc) of phospholipids. The phase transition temperature is one of the most important factors affecting the morphology of liposomes: when the temperature is lower than the phase transition temperature of phospholipids, the arrangement of phospholipids in liposomes is relatively compact, and the state of liposomes is stable; when the ambient temperature is higher than At the phospholipid phase transition temperature, the phospholipids are loosely arranged, and the fluidity within and between liposomes increases. In addition, physical methods such as mechanical extrusion, centrifugation or passing through a gel chromatography column can also better control the particle size of liposomes.

发明内容SUMMARY OF THE INVENTION

针对上述问题，本发明的目的在于提供一种小粒径、高稳定性多室及单室脂质体的同步制备方法，采用所述制备方法制得的多室和单室脂质体粒径均一、表面光滑、分散性良好，且具有良好的储存稳定性。In view of the above-mentioned problems, the object of the present invention is to provide a method for the simultaneous preparation of multi-chambered and unilamellar liposomes with small particle size and high stability, and the particle diameters of multi-chambered and unilamellar liposomes obtained by the preparation method Uniform, smooth surface, good dispersibility, and good storage stability.

为了实现上述目的，本发明采用的具体方案为：In order to achieve the above object, the concrete scheme adopted in the present invention is:

一种小粒径、高稳定性多室及单室脂质体的同步制备方法，包括以下步骤：分别称取大豆卵磷脂、胆固醇和维生素C于500 mL圆底烧瓶中，加入三氯甲烷溶解，再加入玻璃珠于旋转蒸发仪上旋蒸至溶剂挥发完毕，此时圆底烧瓶壁上形成一层均匀薄膜；将圆底烧瓶置于真空干燥箱中干燥过夜；取出圆底烧瓶后，加入浓度0.1M的磷酸盐缓冲溶液60-90 mL进行水合，控制水合温度为35-65℃，氮气环境下涡旋2 h，置于超声仪中超声处理30 min；10000-20000rpm离心，取上清液，用0.22 μm滤膜过滤，于4 ℃冷藏、备用；A method for synchronously preparing multi-chamber and single-chamber liposomes with small particle size and high stability, comprising the following steps: respectively weighing soybean lecithin, cholesterol and vitamin C in a 500 mL round-bottomed flask, adding chloroform to dissolve , then add glass beads and rotate on a rotary evaporator until the solvent evaporates, at this time a uniform film is formed on the wall of the round-bottomed flask; the round-bottomed flask is placed in a vacuum drying oven to dry overnight; after taking out the round-bottomed flask, add 60-90 mL of phosphate buffer solution with a concentration of 0.1M was hydrated, the hydration temperature was controlled at 35-65 °C, vortexed for 2 h in a nitrogen environment, placed in a sonicator for sonication for 30 min; centrifuged at 10000-20000 rpm, and the supernatant was taken The solution was filtered with a 0.22 μm filter membrane, and refrigerated at 4 °C for later use;

所述大豆卵磷脂和胆固醇的总质量为0.11g、质量比为8:1-12:1；所述维生素C的质量为大豆卵磷脂的1%。The total mass of the soybean lecithin and cholesterol is 0.11 g, and the mass ratio is 8:1-12:1; the mass of the vitamin C is 1% of the soybean lecithin.

作为对上述方案的进一步优化，所述大豆卵磷脂和胆固醇的质量比为12:1。As a further optimization of the above scheme, the mass ratio of soybean lecithin and cholesterol is 12:1.

作为对上述方案的进一步优化，所述磷酸缓冲液的用量为80 mL。As a further optimization of the above scheme, the amount of the phosphate buffer was 80 mL.

作为对上述方案的进一步优化，所述离心的转速为20000rpm；As a further optimization to the above scheme, the rotating speed of the centrifugation is 20000rpm;

作为对上述方案的进一步优化，所述水合温度为55℃。As a further optimization of the above scheme, the hydration temperature is 55°C.

有益效果：Beneficial effects:

本发明以脂质体粒径为控制对象，对影响粒径的工艺条件进行了优化，采用改良薄膜分散法制备了粒径约110 nm的多室脂质体，并通过上调水合温度55℃，实现了多室脂质体向单室脂质体的转变，并利用动态光散射（DLS）和透射电子显微镜（TEM）技术检测了多室和单室脂质体的分散性和形态学特征。该制备工艺简单，可同时实现多室脂质体和单室脂质体的制备，制得的多室和单室脂质体粒径均一、表面光滑、分散性良好，且具有良好的储存稳定性。The invention takes the particle size of the liposome as the control object, and optimizes the process conditions that affect the particle size. The multi-lamellar liposome with a particle size of about 110 nm is prepared by the improved film dispersion method, and the hydration temperature is increased by 55 ° C. The conversion of multilamellar liposomes to unilamellar liposomes was achieved, and the dispersion and morphological characteristics of multilamellar and unilamellar liposomes were examined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The preparation process is simple, the preparation of multi-chamber liposomes and unilamellar liposomes can be realized at the same time, and the prepared multi-chamber and unilamellar liposomes have uniform particle size, smooth surface, good dispersibility and good storage stability. sex.

附图说明Description of drawings

图1是多室脂质体的粒径分布图；Fig. 1 is the particle size distribution diagram of multilamellar liposome;

图2是单室脂质体的粒径分布图；Fig. 2 is the particle size distribution figure of unilamellar liposome;

图3是多室和单室脂质体的TEM图；其中，A、B：多室脂质体；C、D：单室脂质体。Figure 3 is a TEM image of multilamellar and unilamellar liposomes; wherein, A, B: multilamellar liposomes; C, D: unilamellar liposomes.

具体实施方式Detailed ways

下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

1 实验部分1 Experimental part

1.1 试剂和仪器1.1 Reagents and instruments

试剂：胆固醇购自西安化学试剂厂；大豆卵磷脂为市售食品级，购自上海金穗生物科技有限公司；维生素C购自天津巴斯夫化工有限公司；三氯甲烷、无水乙醇、乙醚均为市售分析纯试剂；实验室用水购自中国娃哈哈集团有限公司，玻璃珠购自南京奥多福尼生物有限公司。Reagents: cholesterol was purchased from Xi'an Chemical Reagent Factory; soybean lecithin was commercially available food grade, purchased from Shanghai Jinsui Biotechnology Co., Ltd.; vitamin C was purchased from Tianjin BASF Chemical Co., Ltd.; chloroform, absolute ethanol, and ether were Commercially available analytical reagents; laboratory water was purchased from China Wahaha Group Co., Ltd., and glass beads were purchased from Nanjing Odofoni Biological Co., Ltd.

仪器：JEM-2100(UHR)型高分辨透射电子显微镜(JEOL公司)，JEM 1200EX型透射电子显微镜（JEOL公司），Nano-ZS ZEN3600型粒径电位仪（Malvern Instrument），TGL离心机-16G（上海安亭科学仪器厂），SZ-93型自动双显纯化水蒸馏器（上海精密科学仪器有限公司），RE-52型旋转蒸发仪（郑州凯鹏实验仪器有限公司）。Instruments: JEM-2100 (UHR) high-resolution transmission electron microscope (JEOL), JEM 1200EX transmission electron microscope (JEOL), Nano-ZS ZEN3600 particle size potentiometer (Malvern Instrument), TGL centrifuge-16G ( Shanghai Anting Scientific Instrument Factory), SZ-93 Automatic Double Display Purified Water Distiller (Shanghai Precision Scientific Instrument Co., Ltd.), RE-52 Rotary Evaporator (Zhengzhou Kai Peng Experimental Instrument Co., Ltd.).

1.2 脂质体的制备方法1.2 Preparation of liposomes

本实验脂质体的制备是在传统薄膜分散法的基础上进行工艺改进，以粒径为考察对象。称取适量大豆卵磷脂、胆固醇和维生素C于500 mL圆底烧瓶中，加入适量三氯甲烷溶解，再加入玻璃珠于旋转蒸发仪上旋蒸至溶剂挥发完毕。此时圆底烧瓶壁上形成一层均匀薄膜。将圆底烧瓶置于真空干燥箱中干燥过夜。取出圆底烧瓶后，加入浓度0.1M的磷酸盐缓冲溶液（PBS，pH7.4）适量进行水合，控制水合温度，氮气环境下涡旋2 h，置于超声仪中超声处理30 min。离心，取上清液，用0.22 μm滤膜过滤，于4 ℃冷藏、备用。The preparation of liposomes in this experiment is based on the traditional thin film dispersion method, and the process is improved, and the particle size is the object of investigation. Weigh an appropriate amount of soybean lecithin, cholesterol and vitamin C into a 500 mL round-bottom flask, add an appropriate amount of chloroform to dissolve, and then add glass beads and spin on a rotary evaporator until the solvent evaporates. At this time, a uniform film was formed on the wall of the round-bottomed flask. The round bottom flask was placed in a vacuum oven to dry overnight. After taking out the round-bottomed flask, an appropriate amount of phosphate buffer solution (PBS, pH 7.4) with a concentration of 0.1 M was added for hydration, the hydration temperature was controlled, vortexed for 2 h under nitrogen environment, and placed in a sonicator for sonication for 30 min. Centrifuge, take the supernatant, filter through a 0.22 μm filter, and store at 4 °C for later use.

所述大豆卵磷脂和胆固醇的总质量为0.11g；所述维生素C的质量为大豆卵磷脂的1%。The total mass of the soybean lecithin and cholesterol is 0.11 g; the mass of the vitamin C is 1% of the soybean lecithin.

1.3 制备工艺优化1.3 Preparation process optimization

利用单因素方法对卵磷脂与胆固醇的配比、水合时PBS的加入量、离心转速以及水合温度等制备工艺进行优化。这些工艺条件对脂质体的粒径和稳定性具有较大影响。The single factor method was used to optimize the preparation process such as the ratio of lecithin and cholesterol, the amount of PBS added during hydration, the centrifugal speed and the hydration temperature. These process conditions have a great influence on the particle size and stability of liposomes.

1.3.1 卵磷脂与胆固醇的质量比1.3.1 The mass ratio of lecithin to cholesterol

设置4组不同的卵磷脂与胆固醇的质量比8:1、10:1、12:1、14:1，控制卵磷脂和胆固醇总质量为0.11 g。将上述4组卵磷脂和胆固醇分别与0.001 g维生素C置于500 mL圆底烧瓶中，加入2.5 mL三氯甲烷完全溶解，加入直径为0.1 mm的玻璃珠，置于旋转蒸发仪上旋蒸至溶剂挥发完全，放入真空干燥箱中干燥过夜。取出，再加入70 mL磷酸盐缓冲液35 ℃进行水合，室温涡旋2 h，超声30 min，然后以15 000 r/min转速离心30 min，取上清液过0.22μm滤膜。测脂质体粒径，考察卵磷脂与胆固醇质量比对脂质体粒径的影响。Four groups of different mass ratios of lecithin and cholesterol were set at 8:1, 10:1, 12:1, and 14:1, and the total mass of lecithin and cholesterol was controlled to be 0.11 g. The above four groups of lecithin and cholesterol and 0.001 g of vitamin C were placed in a 500 mL round-bottomed flask, 2.5 mL of chloroform was added to completely dissolve, and glass beads with a diameter of 0.1 mm were added, and placed on a rotary evaporator to evaporate to The solvent was completely evaporated, and it was placed in a vacuum drying oven to dry overnight. Take it out, add 70 mL of phosphate buffer solution at 35 °C for hydration, vortex at room temperature for 2 h, sonicate for 30 min, and then centrifuge at 15 000 r/min for 30 min. The particle size of liposomes was measured, and the effect of the mass ratio of lecithin to cholesterol on the particle size of liposomes was investigated.

1.3.2 水合时PBS用量1.3.2 PBS dosage during hydration

PBS的用量决定着最后脂质体悬液的浓度。本实验将PBS用量分别设为60、70、80mL和90 mL，其他条件不变，对不同PBS用量制得的脂质体进行粒径检测，考察其对脂质体粒径的影响。The amount of PBS used determines the final liposomal suspension concentration. In this experiment, the amount of PBS was set to 60, 70, 80 mL and 90 mL, and other conditions remained unchanged. The particle size of liposomes prepared with different amounts of PBS was tested to investigate its effect on the particle size of liposomes.

1.4.3 离心转速1.4.3 Centrifugation speed

离心可以去除粒径过大的脂质体，使脂质体粒径更为均一，因此离心转速对脂质体粒径具有重要影响。本实验设置4组离心转速10 000、12 500、15 000 r/min和20 000 r/min，考察转速对脂质体粒径和分散状态的影响。Centrifugation can remove liposomes with excessive particle size and make the liposome particle size more uniform, so the centrifugal speed has an important influence on the liposome particle size. In this experiment, four groups of centrifugal speeds of 10 000, 12 500, 15 000 r/min and 20 000 r/min were set up to investigate the effect of rotational speed on the particle size and dispersion state of liposomes.

1.3.4 水合温度1.3.4 Hydration temperature

在脂质体制备过程中引入温度控制变量，将水合时的温度分别控制在35、45、55℃和65 ℃，其他条件不变，考察水合温度对于脂质体粒径和分散状态的影响。Temperature control variables were introduced in the liposome preparation process, and the temperature during hydration was controlled at 35, 45, 55 °C and 65 °C, respectively, and other conditions remained unchanged. The influence of hydration temperature on the particle size and dispersion state of liposomes was investigated.

1.3.5 重现性实验1.3.5 Reproducibility experiment

综合以上的工艺优化方案，在最佳工艺条件下，制备3批脂质体，采用动态光散射法（DLS）测定3组脂质体的粒径，考察制备工艺的重现性。Based on the above process optimization plan, under the best process conditions, three batches of liposomes were prepared, and the particle size of the three groups of liposomes was determined by dynamic light scattering (DLS) to investigate the reproducibility of the preparation process.

1.4 脂质体粒径、分散性的测定1.4 Determination of liposome particle size and dispersibility

室温条件下，取适量脂质体悬液，用PBS稀释至适当浓度。将稀释后的1 mL脂质体溶液置于样品皿中，放入激光粒度分析仪样品池中，测其粒径和多分散性指数（PDI）值，每个样品测定3次。At room temperature, take an appropriate amount of liposome suspension and dilute it with PBS to an appropriate concentration.Place 1 mL of the diluted liposome solution in a sample dish, put it into the sample cell of a laser particle size analyzer, and measure its particle size and polydispersity index (PDI) value, three times for each sample.

1.5 脂质体形态观察1.5 Observation of liposome morphology

用移液枪吸取10 μL稀释后的脂质体悬液滴于铜网上，静置15 min，自然晾干，于透射电镜下观察脂质体的形态。Pipette 10 μL of the diluted liposome suspension onto a copper mesh, let it stand for 15 min, and then dry it naturally. The morphology of the liposomes was observed under a transmission electron microscope.

1.6 脂质体储存稳定性考察1.6 Investigation of storage stability of liposomes

将脂质体悬液置于4 ℃，间隔一定时间观察脂质体颜色变化以及是否有沉淀产生，并分别于1d、6d 、30d和60 d测其粒径和PDI值，考察脂质体的存储稳定性。The liposome suspension was placed at 4 °C, and the color changes of liposomes and whether there was any precipitation were observed at certain intervals, and the particle size and PDI value were measured at 1d, 6d, 30d and 60d, respectively, and the liposomes were investigated. Storage stability.

2 实验结果2 Experimental results

2.1 脂质体制备的工艺优化2.1 Process optimization of liposome preparation

2.1.1 卵磷脂与脂质体之比2.1.1 Ratio of lecithin to liposome

不同质量比卵磷脂与胆固醇制得的脂质体粒径、PDI值汇总结果如表1所示。从表1中可以看出，随着卵磷脂与胆固醇的比例从8:1增加到12:1时，脂质体粒径逐渐减小，当二者比例为12:1时，此时粒径最小，为~137 nm，PDI为0.212，分散性良好。但随着卵磷脂的比例继续增加到14:1，脂质体粒径增加到~147nm，PDI为0.267，脂质体分散性变差。Table 1 shows the summary results of particle size and PDI value of liposomes prepared with different mass ratios of lecithin and cholesterol. It can be seen from Table 1 that as the ratio of lecithin to cholesterol increases from 8:1 to 12:1, the particle size of liposomes gradually decreases. When the ratio of the two is 12:1, the particle size The smallest, at ~137 nm, has a PDI of 0.212 and good dispersion. But as the ratio of lecithin continued to increase to 14:1, the liposome particle size increased to ~147 nm, the PDI was 0.267, and the liposome dispersibility became worse.

表1 卵磷脂与胆固醇质量比对脂质体粒径的影响Table 1 Effect of mass ratio of lecithin and cholesterol on liposome particle size

2.1.2 PBS用量2.1.2 Dosage of PBS

将卵磷脂与胆固醇质量比固定为12:1，改变水合时PBS用量，得到的脂质体粒径和PDI的结果如表2所示。从表2中可以看出，当PBS用量为80 mL时，得到的脂质体的粒径为~121 nm， PDI为0.198。后续实验将PBS用量设定为80 mL。The mass ratio of lecithin to cholesterol was fixed at 12:1, and the amount of PBS during hydration was changed. The obtained liposome particle size and PDI results are shown in Table 2. As can be seen from Table 2, when the amount of PBS was 80 mL, the obtained liposomes had a particle size of ~121 nm and a PDI of 0.198. Subsequent experiments set the amount of PBS to 80 mL.

表2 PBS用量对粒径影响结果Table 2 Effect of PBS dosage on particle size

2.1.3 离心转速2.1.3 Centrifugation speed

将卵磷脂与胆固醇质量比固定为12:1，水合时PBS用量固定为80 mL，改变离心转速，得到的脂质体粒径和PDI 结果如表3所示。从表3可以看出，当离心转速为20000 r/min时，得到的脂质体粒径最小，为~114 nm，PDI值为0.214。The mass ratio of lecithin to cholesterol was fixed at 12:1, the amount of PBS during hydration was fixed at 80 mL, and the centrifugal speed was changed. The obtained liposome particle size and PDI results are shown in Table 3. It can be seen from Table 3 that when the centrifugation speed is 20000 r/min, the obtained liposomes have the smallest particle size, which is ~114 nm, and the PDI value is 0.214.

表3 离心转速对粒径影响结果Table 3 Effect of centrifugal speed on particle size

2.1.4 水合温度2.1.4 Hydration temperature

将卵磷脂与胆固醇质量比固定为12:1，水合时PBS用量固定为80 mL，离心转速设定为20 000 r/min，改变水合温度，得到的脂质体粒径、PDI结果如表4所示。由于水合温度对脂质体的形态具有重要影响，因此，在优化水合温度时利用TEM对脂质体的微观形貌进行了考察。从表4中可以看出，水合温度对脂质体的粒径影响不大，但对脂质体的形貌影响较大，当温度上调至55 ℃（卵磷脂的相变温度）时，脂质体从多室转变为单室。后续多室脂质体制备时的水合温度控制在35 ℃，单室脂质体的水合温度控制在55 ℃。The mass ratio of lecithin to cholesterol was fixed at 12:1, the amount of PBS during hydration was fixed at 80 mL, the centrifugal speed was set at 20 000 r/min, and the hydration temperature was changed. The obtained liposome particle size and PDI results are shown in Table 4. shown. Since the hydration temperature has an important influence on the morphology of the liposomes, the microscopic morphology of the liposomes was investigated by TEM when the hydration temperature was optimized. It can be seen from Table 4 that the hydration temperature has little effect on the particle size of liposomes, but has a greater effect on the morphology of liposomes. When the temperature is increased to 55 °C (the phase transition temperature of lecithin), the The plastid transforms from multicompartment to unicompartment. The hydration temperature of the subsequent multilamellar liposome preparation was controlled at 35 °C, and the hydration temperature of the unilamellar liposome was controlled at 55 °C.

表4 水合温度对于脂质体形态的影响Table 4 Effect of hydration temperature on liposome morphology

2.1.5 方法的重现性2.1.5 Reproducibility of the method

在多室和单室脂质体的最佳制备工艺条件下，分别进行三次平行实验，结果如表5和表6所示。其中，多室脂质体的平均粒径为~109 nm，单室脂质体的平均粒径为~108 nm，且两种形态的脂质体的PDI值均小于0.2，分散性良好。据此可以说明本文制备脂质体的工艺方法重现性较好。Under the optimal preparation process conditions of multi- and unilamellar liposomes, three parallel experiments were carried out respectively, and the results are shown in Table 5 and Table 6. Among them, the average particle size of multilamellar liposomes is ~109 nm, and the average particle size of unilamellar liposomes is ~108 nm, and the PDI values of the two forms of liposomes are both less than 0.2, showing good dispersibility. According to this, it can be shown that the process method for preparing liposomes in this paper has good reproducibility.

表5 多室脂质体重现性实验结果Table 5 Reproducibility experimental results of multilamellar liposomes

多室multi-chamber粒径/nmParticle size/nmPDIPDI11114±5114±50.1900.19022107±1107±10.1880.18833107±2107±20.1850.185均值mean109±4109±40.1880.188

表6 单室脂质体重现性实验结果Table 6 Reproducibility experimental results of unilamellar liposomes

单室single room粒径/nmParticle size/nmPDIPDI11110±4110±40.1740.17422108±2108±20.1690.16933106±1106±10.1600.160均值mean108±2108±20.1680.168

2.2脂质体粒径分布2.2 Liposome particle size distribution

最佳工艺条件下制备的多室和单室脂质体的DLS图谱如图1-2所示。多室（MVs）和单室（UVs）脂质体DLS图谱均呈现尖锐单峰，粒径分别为~110和~106 nm，PDI分别为0.185和0.167，表示脂质体粒径大小均一、分散性良好。The DLS profiles of multi- and unilamellar liposomes prepared under optimal process conditions are shown in Figures 1-2. The DLS spectra of multi-chambered (MVs) and single-chambered (UVs) liposomes both showed sharp single peaks, with particle sizes of ~110 and ~106 nm, and PDI of 0.185 and 0.167, respectively, indicating that the liposomes were uniform in size and dispersed in size. Sex is good.

2. 3脂质体的微观形貌2.3 Microscopic morphology of liposomes

观察最佳工艺条件下制得脂质体的外观形貌，如图3所示。从图3A和3B中可以很清晰的看到，视野中出现一个大囊泡里面包裹数个小囊泡的脂质体，显示出典型多室脂质体的特征^[11]。图3C和3D则为单室囊泡形貌，囊泡表面光滑。The appearance and morphology of the liposomes prepared under the optimal process conditions were observed, as shown in Figure 3. It can be clearly seen from Figures 3A and 3B that a large vesicle encapsulates liposomes with several small vesicles in the field of view, showing the characteristics of typical multilamellar liposomes^[11] . Figures 3C and 3D show the morphology of unilamellar vesicles with smooth vesicle surfaces.

2.4脂质体的外观及储存稳定性2.4 Appearance and storage stability of liposomes

从外观来看，制得的多室和单室脂质体悬液均为无色透明且有乳光的液体，4 ℃下存放60 d后依然清澈无沉淀产生。将5组最佳工艺下制备的多室和单室脂质体放入4 ℃环境中，分别在1、6、30 d和60 d测其粒径，结果如表7和表8所示。可以看出，30 d后多室和单室脂质体粒径仍无明显变化。60 d后脂质体粒径略有增加，但增加幅度不大，表明该工艺条件下制得的多室和单室脂质体具有良好的存储稳定性。In terms of appearance, the prepared multi- and unilamellar liposome suspensions are colorless, transparent and opalescent liquids, which are still clear and free of precipitation after being stored at 4 °C for 60 d. The multi- and unilamellar liposomes prepared under the five optimal processes were placed in a 4 ℃ environment, and their particle sizes were measured at 1, 6, 30 d and 60 d, respectively. The results are shown in Tables 7 and 8. It can be seen that the particle size of multi- and unilamellar liposomes remained unchanged after 30 days. After 60 days, the particle size of liposomes increased slightly, but the increase was not large, indicating that the multi- and unilamellar liposomes prepared under this process had good storage stability.

表7 多室脂质体稳定性实验结果Table 7 Stability test results of multilamellar liposomes

多室multi-chamber时间/dtime/d粒径/nmParticle size/nm1111108±1108±12266111±1111±1333030113±3113±3446060114±2114±2

表8 单室脂质体稳定性实验结果Table 8 Stability test results of unilamellar liposomes

单室single room时间/dtime/d粒径/nmParticle size/nm1111106±1106±12266108±1108±1333030109±3109±3446060112±2112±2

本发明对卵磷脂与胆固醇质量比、水合时PBS的用量、离心转速和水合温度4个脂质体制备过程中的重要变量进行工艺优化，以达到制备粒径较小、分散性良好且稳定性良好的脂质体的目的。脂质体制备中胆固醇的加入有利于增大膜的刚性和减小粒径，使卵磷脂双分子层结构更稳固。然而当胆固醇的添加量超过了双分子层的容纳限度后，反而会造成膜结构的破坏，使脂质体失去稳定性。如表1所示，随着卵磷脂与胆固醇的比例从8:1增加到12:1时，脂质体粒径逐渐减小，当二者比例为12:1时，此时粒径最小，为~137 nm，PDI为0.212，分散性良好。但随着卵磷脂的比例继续增加到14:1，脂质体粒径增加到~147nm，PDI为0.267，脂质体分散性变差。因此，合适的卵磷脂、胆固醇质量比是脂质体稳定的必要条件。本发明发现，卵磷脂和胆固醇的质量比为12:1时，脂质体的粒径较小、稳定性较好。The present invention optimizes the process of four important variables in the preparation process of liposomes, including the mass ratio of lecithin and cholesterol, the amount of PBS during hydration, the centrifugal speed and the hydration temperature, so as to achieve smaller particle size, good dispersion and stability. Good for liposome purpose. The addition of cholesterol in the preparation of liposomes is beneficial to increase the rigidity of the membrane and reduce the particle size, so that the lecithin bilayer structure is more stable. However, when the amount of cholesterol added exceeds the accommodating limit of the bilayer, it will cause the destruction of the membrane structure and make the liposome lose its stability. As shown in Table 1, as the ratio of lecithin to cholesterol increased from 8:1 to 12:1, the particle size of liposomes gradually decreased. When the ratio of the two was 12:1, the particle size was the smallest. is ~137 nm, the PDI is 0.212, and the dispersion is good. But as the ratio of lecithin continued to increase to 14:1, the liposome particle size increased to ~147 nm, the PDI was 0.267, and the liposome dispersibility became worse. Therefore, a suitable mass ratio of lecithin and cholesterol is a necessary condition for the stability of liposomes. It is found in the present invention that when the mass ratio of lecithin and cholesterol is 12:1, the particle size of the liposome is smaller and the stability is better.

水合时PBS的用量对脂质体的粒径和稳定性也有重要的影响。PBS用量过少，则脂质体悬液浓度过大，脂质体之间容易相互融合。因此，合适的PBS用量也是制备性能良好脂质体所必须考虑的因素。离心转速是影响脂质体粒径和均一性的重要因素。结果如表3所示，随着离心转速的增加，脂质体粒径随之减小，当转速达到20 000 r/min时，所得的脂质体粒径最小。The amount of PBS used during hydration also had an important effect on the particle size and stability of the liposomes. If the amount of PBS is too small, the concentration of the liposome suspension will be too large, and the liposomes will easily fuse with each other. Therefore, the appropriate amount of PBS is also a factor that must be considered in the preparation of liposomes with good performance. Centrifugation speed is an important factor affecting the particle size and homogeneity of liposomes. The results are shown in Table 3. With the increase of the centrifugal speed, the particle size of the liposomes decreased. When the rotation speed reached 20 000 r/min, the obtained liposome particle size was the smallest.

为了得到小粒径的单室脂质体，本发明在前期多室脂质体制备的最佳工艺条件下，将水合温度作为控制变量，上调水合温度至磷脂的相变温度55 ℃，干燥时加入直径为0.1 mm的玻璃珠，后续放在超声仪中超声处理30 min。结果显示，当水合温度大于55℃时，制得的脂质体从多室转变为单室脂质体，并且粒径成功控制在110 nm左右。本发明仅利用水合温度的控制，即实现了多室和单室脂质体的同时制备。In order to obtain unilamellar liposomes with small particle size, under the optimal process conditions for the preparation of multilamellar liposomes in the early stage, the hydration temperature is used as a control variable, and the hydration temperature is raised to the phase transition temperature of phospholipids of 55 °C. Glass beads with a diameter of 0.1 mm were added, and then placed in a sonicator for sonication for 30 min. The results showed that when the hydration temperature was greater than 55 °C, the prepared liposomes changed from multi-chambered to unilamellar liposomes, and the particle size was successfully controlled at about 110 nm. The present invention realizes the simultaneous preparation of multi-chambered and unilamellar liposomes only by controlling the hydration temperature.

脂质体的粒径对于脂质体的体内生物学效应影响较大，当粒径小于20 nm时，脂质体容易被肾脏清除排出体外，体内半衰期短，不容易在靶细胞积累，但脂质体粒径过大则容易被体内巨噬细胞识别并吞噬，同样缩短体内的半衰期。粒径在100 nm左右的脂质体具有较好的体内稳定性和肿瘤部位蓄积的能力。因此，本发明制备的分散均一、稳定性良好，粒径约110 nm的多室和单室脂质体，预期可以实现较好的体内生物学效应。The particle size of liposomes has a great influence on the in vivo biological effects of liposomes. When the particle size is less than 20 nm, liposomes are easily cleared by the kidneys and excreted from the body, and the half-life in vivo is short, and it is not easy to accumulate in target cells. If the particle size of plastids is too large, it is easy to be recognized and phagocytosed by macrophages in vivo, which also shortens the half-life in vivo. Liposomes with a particle size of about 100 nm have better in vivo stability and the ability to accumulate in tumor sites. Therefore, the multi-chambered and unilamellar liposomes with uniform dispersion and good stability and particle size of about 110 nm prepared by the present invention are expected to achieve better biological effects in vivo.

需要说明的是，以上所述的实施方案应理解为说明性的，而非限制本发明的保护范围，本发明的保护范围以权利要求书为准。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对本发明作出的一些非本质的改进和调整仍属于本发明的保护范围。It should be noted that the above-mentioned embodiments should be construed as illustrative rather than limiting the protection scope of the present invention, which is subject to the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some non-essential improvements and adjustments made to the present invention still belong to the protection scope of the present invention.

Claims (2)

1. A synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability comprises the following steps: respectively weighing soybean lecithin, cholesterol and vitamin C, adding trichloromethane into a 500 mL round-bottom flask for dissolving, adding glass beads into the flask, and performing rotary evaporation on the glass beads in a rotary evaporator until the solvent is completely volatilized, wherein a layer of uniform film is formed on the wall of the round-bottom flask; putting the round-bottom flask into a vacuum drying oven to dry overnight; taking out the round-bottom flask, adding 80 mL of phosphate buffer solution with the concentration of 0.1M for hydration, controlling the hydration temperature to be 35-65 ℃, performing vortex for 2 h in a nitrogen environment, and performing ultrasonic treatment for 30 min in an ultrasonic instrument; centrifuging at 20000rpm, collecting supernatant, filtering with 0.22 μm filter membrane, and refrigerating at 4 deg.C;

the total mass of the soybean lecithin and the cholesterol is 0.11g, and the mass ratio is 12; the mass of the vitamin C is 1 percent of that of the soybean lecithin.

2. The method of claim 1, wherein: the hydration temperature was 55 ℃.

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