CN112545994A

CN112545994A - Synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability

Info

Publication number: CN112545994A
Application number: CN202011297330.3A
Authority: CN
Inventors: 梁菊; 吴文澜; 郭润发; 孙乾坤; 何佳彧
Original assignee: Henan University of Science and Technology
Current assignee: Henan University of Science and Technology
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-03-26
Anticipated expiration: 2040-11-19
Also published as: CN112545994B

Abstract

The invention relates to a synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability, which belongs to the field of chemical industry and pharmacy, takes liposome particle size as a control object, optimizes process conditions influencing the particle size by utilizing a single-factor experiment, prepares the multi-chamber liposome with the particle size of about 110 nm by adopting an improved film dispersion method, realizes the conversion from the multi-chamber liposome to the single-chamber liposome by adjusting the hydration temperature to 55 ℃, and detects the dispersibility and morphological characteristics of the multi-chamber and single-chamber liposome by utilizing Dynamic Light Scattering (DLS) and Transmission Electron Microscope (TEM) technologies. The preparation process is simple, can simultaneously realize the preparation of the multi-chamber liposome and the single-chamber liposome, and the prepared multi-chamber liposome and single-chamber liposome have uniform particle size, smooth surface, good dispersibility and good storage stability.

Description

Synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability

Technical Field

The invention belongs to the technical field of chemical industry and pharmacy, and particularly relates to a synchronous preparation method of multi-chamber and single-chamber liposomes.

Background

Liposomes (liposomes) are bilayer lipid vesicles composed of amphiphilic molecules such as soybean lecithin and cholesterol, and have particle diameters varying from 10 nm to 2 μm. Liposomes can be classified into the following 3 types according to the difference in particle size and number of layers: unilamellar liposomes having a particle size of 10 to 100 nm are called Small Unilamellar Vesicles (SUVs); unilamellar liposomes with a particle size of 100 nm to 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 having a small particle diameter are relatively rare. The particle size of liposomes is a key parameter in determining their stability and in vivo applications. The multi-chamber liposome is formed by overlapping a plurality of concentric lipid round vesicles, the number of lipid layers is large, the entrapment rate of lipid-soluble medicines in gaps among lipid layers is high, but the distance between layers is small, membrane fusion is easy to occur, and the particle size of the liposome is large and unstable. The problems of poor stability, uneven particle size and the like of the unilamellar liposomes with overlarge particle sizes exist. Generally, liposomes are more stable as their particle size is smaller, and liposomes having a particle size of about 100 nm are highly preferred because they have a good EPR Effect (Enhanced Permeability and retentivity Effect) at a tumor site. In addition, the particle size of the liposome also directly affects whether the carrier can smoothly enter tissues and cells to play a role.

Factors that influence liposome particle size include the type of phospholipid used in preparing the liposome, the ratio of phospholipid to cholesterol, and the phase transition temperature (Tc) of the phospholipid. Phase transition temperature is one of the most important factors affecting liposome morphology: when the temperature is lower than the phase transition temperature of the phospholipids, the arrangement of the phospholipids in the liposome is compact, and the liposome is stable; when the environmental temperature is higher than the phase transition temperature of the phospholipids, the phospholipids are loosely arranged, and the fluidity inside the liposomes and among the liposomes is increased. In addition, the particle size of the liposome can be well controlled by physical methods such as mechanical extrusion, centrifugation or gel chromatography column.

Disclosure of Invention

In view of the above problems, the present invention provides a method for synchronously preparing small-particle-size and high-stability multi-chamber and single-chamber liposomes, wherein the multi-chamber and single-chamber liposomes prepared by the method have uniform particle size, smooth surface, good dispersibility, and good storage stability.

In order to achieve the purpose, the invention adopts the specific scheme that:

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 60-90 mL of phosphate buffer solution with the concentration of 0.1M for hydration, controlling the hydration temperature to be 35-65 ℃, carrying out vortex for 2 h in a nitrogen environment, and placing the mixture in an ultrasonic instrument for ultrasonic treatment for 30 min; 10000-;

the total mass of the soybean lecithin and the cholesterol is 0.11g, and the mass ratio is 8:1-12: 1; the mass of the vitamin C is 1 percent of that of the soybean lecithin.

As a further optimization of the scheme, the mass ratio of the soybean lecithin to the cholesterol is 12: 1.

As a further optimization of the above protocol, the amount of phosphate buffer used was 80 mL.

As a further optimization of the scheme, the rotation speed of the centrifugation is 20000 rpm;

as a further optimization of the above protocol, the hydration temperature is 55 ℃.

Has the advantages that:

the invention takes liposome particle size as a control object, optimizes the process conditions influencing the particle size, adopts an improved film dispersion method to prepare the multi-chamber liposome with the particle size of about 110 nm, realizes the conversion from the multi-chamber liposome to the single-chamber liposome by adjusting the hydration temperature to 55 ℃, and detects the dispersibility and morphological characteristics of the multi-chamber and single-chamber liposome by utilizing Dynamic Light Scattering (DLS) and Transmission Electron Microscope (TEM) technologies. The preparation process is simple, can simultaneously realize the preparation of the multi-chamber liposome and the single-chamber liposome, and the prepared multi-chamber liposome and single-chamber liposome have uniform particle size, smooth surface, good dispersibility and good storage stability.

Drawings

FIG. 1 is a particle size distribution plot of a multilamellar liposome;

FIG. 2 is a particle size distribution plot of a unilamellar liposome;

FIG. 3 is a TEM image of multi-compartmental and single-compartmental liposomes; wherein, A, B: a multilamellar liposome; C. d: unilamellar liposomes.

Detailed Description

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 experimental part

1.1 reagents and instruments

Reagent: cholesterol was purchased from sienna chemical reagent factory; the soybean lecithin is a commercially available food grade and is purchased from Shanghai Jinsui Biotech limited; vitamin C was purchased from Tianjin Pasteur chemical Co., Ltd; the trichloromethane, the absolute ethyl alcohol and the diethyl ether are all commercially available analytical pure reagents; laboratory water was purchased from Wahaha group, Inc. of China, and glass beads were purchased from Odofny, Bio, Inc. of Nanjing.

The instrument comprises the following steps: JEM-2100(UHR) type high-resolution transmission electron microscope (JEOL corporation), JEM 1200EX type transmission electron microscope (JEOL corporation), Nano-ZS ZEN3600 type particle size potentiometer (Malvern Instrument), TGL centrifuge-16G (Shanghai Tingning scientific instruments factory), SZ-93 type automatic double-display purified water distiller (Shanghai precision scientific instruments Co., Ltd.), RE-52 type rotary evaporator (Zhengzhou Kaepeng laboratory instruments Co., Ltd.).

1.2 preparation of liposomes

The preparation of the experimental liposome is carried out by improving the process on the basis of the traditional film dispersion method, and the particle size is taken as an investigation object. Weighing a proper amount of soybean lecithin, cholesterol and vitamin C, adding a proper amount of chloroform into a 500 mL round-bottom flask for dissolving, adding glass beads, and performing rotary evaporation on a rotary evaporator until the solvent is completely volatilized. At this time, a uniform film is formed on the wall of the round-bottom flask. The round bottom flask was placed in a vacuum oven to dry overnight. After taking out the round-bottom flask, adding a proper amount of phosphate buffer solution (PBS, pH7.4) with the concentration of 0.1M for hydration, controlling the hydration temperature, swirling for 2 h in a nitrogen environment, and placing the mixture in an ultrasonic instrument for ultrasonic treatment for 30 min. Centrifuging, 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 0.11 g; the mass of the vitamin C is 1 percent of that of the soybean lecithin.

1.3 optimization of the preparation Process

The preparation processes such as the ratio of lecithin to cholesterol, the addition of PBS during hydration, the centrifugal speed, the hydration temperature and the like are optimized by a single-factor method. These process conditions have a large influence on the particle size and stability of the liposomes.

1.3.1 weight ratio of lecithin to Cholesterol

The mass ratio of lecithin to cholesterol of 4 groups of different groups is set to be 8:1, 10:1, 12:1 and 14:1, and the total mass of the lecithin and the cholesterol is controlled to be 0.11 g. Placing the above 4 groups of lecithin and cholesterol respectively with 0.001 g vitamin C in a 500 mL round bottom flask, adding 2.5 mL chloroform for complete dissolution, adding glass beads with diameter of 0.1 mm, placing in a rotary evaporator for rotary evaporation until the solvent is completely volatilized, and placing in a vacuum drying oven for drying overnight. Taking out, adding 70 mL phosphate buffer solution, hydrating at 35 deg.C, vortexing at room temperature for 2 h, sonicating for 30 min, centrifuging at 15000 r/min for 30 min, and filtering the supernatant with 0.22 μm filter membrane. And measuring the liposome particle size, and inspecting the influence of the mass ratio of lecithin and cholesterol on the liposome particle size.

1.3.2 amounts of PBS on hydration

The amount of PBS used determines the concentration of the final liposome suspension. In the experiment, the PBS dosage is respectively set to be 60 mL, 70 mL, 80 mL and 90 mL, other conditions are not changed, the particle size of the liposome prepared by different PBS dosages is detected, and the influence of the particle size of the liposome is examined.

1.4.3 centrifugal rotational speed

The centrifugation can remove the liposome with overlarge particle size, so that the particle size of the liposome is more uniform, and the centrifugation rotating speed has important influence on the particle size of the liposome. 4 groups of centrifugal rotating speeds 10000, 12500, 15000 r/min and 20000 r/min are set in the experiment, and the influence of the rotating speeds on the particle size and the dispersion state of the liposome is examined.

1.3.4 hydration temperature

Introducing temperature control variables in the preparation process of the liposome, controlling the temperatures in hydration to be 35, 45, 55 ℃ and 65 ℃ respectively, keeping other conditions unchanged, and investigating the influence of the hydration temperature on the particle size and the dispersion state of the liposome.

1.3.5 reproducibility test

By combining the process optimization schemes, 3 batches of liposomes are prepared under the optimal process conditions, the particle sizes of 3 groups of liposomes are measured by adopting a dynamic light scattering method (DLS), and the reproducibility of the preparation process is inspected.

1.4 measurement of particle size and dispersibility of liposomes

At room temperature, an appropriate amount of liposome suspension was taken and diluted with PBS to an appropriate concentration. The diluted 1 mL liposome solution was placed in a sample dish and placed in a sample cell of a laser particle size analyzer to measure the particle size and polydispersity index (PDI) values, each sample being measured 3 times.

1.5 Liposome morphology Observation

And (3) sucking 10 mu L of diluted liposome suspension liquid by using a pipette gun, dripping the diluted liposome suspension liquid on a copper net, standing for 15 min, naturally airing, and observing the form of the liposome under a transmission electron microscope.

1.6 Liposome storage stability Studies

And (3) placing the liposome suspension at 4 ℃, observing the color change of the liposome and whether precipitates are generated or not at intervals, and detecting the particle size and the PDI value of the liposome at 1d, 6d, 30d and 60 d respectively to investigate the storage stability of the liposome.

2 results of the experiment

2.1 Process optimization of Liposome preparation

2.1.1 lecithin to Liposome ratio

The particle size and PDI values of the liposomes prepared with lecithin and cholesterol in different mass ratios are summarized in Table 1. As can be seen from Table 1, the liposome particle size gradually decreased as the ratio of lecithin to cholesterol increased from 8:1 to 12:1, and when the ratio of lecithin to cholesterol was 12:1, the particle size was at a minimum of 137 nm, PDI was 0.212, and dispersibility was good. However, as the lecithin ratio continues to increase to 14:1, liposome particle size increases to-147 nm, PDI is 0.267, and liposome dispersibility deteriorates.

TABLE 1 Effect of lecithin to Cholesterol mass ratio on liposome particle size

2.1.2 amounts of PBS

The results of the particle size and PDI of the liposomes obtained with the fixed mass ratio of lecithin to cholesterol of 12:1 and varying the amount of PBS used in the hydration are shown in table 2. As can be seen from Table 2, when the amount of PBS was 80 mL, the particle size of the obtained liposomes was 121 nm and the PDI was 0.198. Subsequent experiments set the PBS usage to 80 mL.

TABLE 2 PBS usage on particle size results

2.1.3 centrifugal rotational speed

The results of particle size and PDI of the liposomes obtained with the fixed mass ratio of lecithin to cholesterol of 12:1 and the fixed amount of PBS used in hydration of 80 mL and the changed centrifuge speed are shown in Table 3. As can be seen from Table 3, when the centrifugal rotation speed is 20000 r/min, the obtained liposome has the smallest particle size of 114 nm and the PDI value of 0.214.

TABLE 3 results of the influence of centrifugal speed on particle size

2.1.4 hydration temperature

The results of the particle size and PDI of the liposomes obtained with the fixed mass ratio of lecithin to cholesterol of 12:1, the fixed amount of PBS during hydration of 80 mL, the centrifuge rotation speed of 20000 r/min and the changed hydration temperature are shown in Table 4. Since hydration temperature has an important effect on liposome morphology, the microscopic morphology of liposomes was investigated using TEM when optimizing hydration temperature. As can be seen from table 4, the hydration temperature has little effect on the particle size of the liposomes, but has a greater effect on the morphology of the liposomes, and when the temperature is increased to 55 ℃ (the phase transition temperature of lecithin), the liposomes are transformed from multi-compartment to single-compartment. The hydration temperature of the subsequent multi-chamber liposome preparation is controlled at 35 ℃, and the hydration temperature of the single-chamber liposome is controlled at 55 ℃.

TABLE 4 Effect of hydration temperature on liposome morphology

2.1.5 reproducibility of the method

In the optimal preparation process conditions for multi-compartment and single-compartment liposomes, three parallel experiments were performed, and the results are shown in tables 5 and 6. Wherein the average particle size of the multi-chamber liposome is 109 nm, the average particle size of the single-chamber liposome is 108 nm, the PDI values of the two forms of liposome are less than 0.2, and the dispersibility is good. Therefore, the process method for preparing the liposome has better reproducibility.

TABLE 5 results of multi-compartment liposome reproducibility experiments

Multi-chamber	Particle size/nm	PDI
				1	114±5	0.190
2	107±1	0.188
			3	107±2	0.185
Mean value	109±4	0.188

TABLE 6 results of single-compartment liposome reproducibility experiments

Single chamber	Particle size/nm	PDI
				1	110±4	0.174
2	108±2	0.169
			3	106±1	0.160
Mean value	108±2	0.168

2.2 Liposome particle size distribution

DLS profiles of multi-compartment and single-compartment liposomes prepared under optimal process conditions are shown in FIGS. 1-2. DLS spectra of multi-compartment (MVs) and single compartment (UVs) liposomes show sharp single peaks, the particle sizes are 110-106 nm respectively, PDI is 0.185-0.167 respectively, and the liposomes are uniform in particle size and good in dispersibility.

2.3 micro-morphology of liposomes

The appearance of the liposomes prepared under the optimal process conditions was observed, as shown in fig. 3. As can be clearly seen in FIGS. 3A and 3B, the field of view shows liposomes with a large vesicle and several small vesicles encapsulated therein, showing the characteristics of typical multilamellar liposomes^[11]. FIGS. 3C and 3D show the morphology of unilamellar vesicles, which are smooth on the surface.

2.4 appearance and storage stability of liposomes

From the appearance, the prepared multi-chamber and single-chamber liposome suspensions are colorless and transparent liquids with opalescence, and are still clear and have no precipitate after being stored for 60 days at 4 ℃. The multi-compartment and single-compartment liposomes prepared by the optimal process of 5 groups were placed in an environment of 4 ℃ and their particle sizes were measured at 1, 6, 30d and 60 d, respectively, and the results are shown in tables 7 and 8. It can be seen that there was no significant change in the particle size of the multi-and unilamellar liposomes after 30 d. After 60 days, the particle size of the liposome is slightly increased, but the increase amplitude is not large, which indicates that the multi-chamber liposome and the single-chamber liposome prepared under the process condition have good storage stability.

TABLE 7 results of multichamber liposome stability experiments

Multi-chamber	Time/d	Particle size/nm
			1	1	108±1
2	6	111±1
			3	30	113±3
4	60	114±2

TABLE 8 results of the stability experiment of the unilamellar liposomes

Single chamber	Time/d	Particle size/nm
			1	1	106±1
2	6	108±1
			3	30	109±3
4	60	112±2

The invention carries out process optimization on the important variables in the preparation process of 4 liposomes including the mass ratio of lecithin to cholesterol, the dosage of PBS during hydration, the centrifugal rotating speed and the hydration temperature, thereby achieving the purpose of preparing the liposome with smaller particle size, good dispersibility and good stability. The addition of cholesterol in the preparation of the liposome is beneficial to increasing the rigidity of the membrane and reducing the particle size, so that the lecithin bilayer structure is more stable. However, when the amount of cholesterol added exceeds the bilayer tolerance limit, the membrane structure is destroyed, and the liposome loses stability. As shown in Table 1, the liposome particle size gradually decreased as the ratio of lecithin to cholesterol increased from 8:1 to 12:1, and when the ratio was 12:1, the particle size was at a minimum of 137 nm and PDI was 0.212, which resulted in good dispersibility. However, as the lecithin ratio continues to increase to 14:1, liposome particle size increases to-147 nm, PDI is 0.267, and liposome dispersibility deteriorates. Therefore, a suitable lecithin to cholesterol mass ratio is a necessary condition for liposome stabilization. The invention discovers that when the mass ratio of the lecithin to the cholesterol is 12:1, the liposome has smaller particle size and better stability.

The amount of PBS used in hydration also has an important effect on the particle size and stability of the liposomes. If the PBS dosage is too small, the liposome suspension concentration is too high, and the liposomes are easy to fuse with each other. Thus, the appropriate amount of PBS is also a factor that must be considered in preparing a well-performing liposome. The centrifugation speed is an important factor affecting the particle size and uniformity of the liposomes. As shown in Table 3, the liposome particle size decreased with increasing centrifugation speed, and the liposome particle size was minimized when the rotation speed reached 20000 r/min.

In order to obtain the single-chamber liposome with small particle size, the invention takes the hydration temperature as a control variable under the optimal process condition of the previous preparation of the multi-chamber liposome, the hydration temperature is adjusted up to 55 ℃ of the phase transition temperature of phospholipid, glass beads with the diameter of 0.1 mm are added during drying, and then the single-chamber liposome is placed in an ultrasonic instrument for ultrasonic treatment for 30 min. The results show that when the hydration temperature is higher than 55 ℃, the prepared liposome is converted from multi-chamber to single-chamber liposome, and the particle size is successfully controlled to be about 110 nm. The invention realizes the simultaneous preparation of multi-chamber and single-chamber liposome only by controlling the hydration temperature.

The liposome has a large influence on the in vivo biological effect of the liposome, when the particle size is less than 20 nm, the liposome is easy to be cleared and discharged out of a body by a kidney, the in vivo half-life period is short, the liposome is not easy to accumulate in target cells, but the liposome is easy to be recognized and phagocytized by in vivo macrophages when the particle size is too large, and the in vivo half-life period is also shortened. Liposomes having a particle size of about 100 nm have good in vivo stability and tumor site accumulation ability. Therefore, the multi-chamber and single-chamber liposome which is uniform in dispersion, good in stability and about 110 nm in particle size and prepared by the invention is expected to realize better in vivo biological effect.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.

Claims

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 60-90 mL of 0.1M phosphate buffer solution for hydration, controlling the hydration temperature to be 35-65 ℃, carrying out vortex for 2 h in a nitrogen environment, and placing the mixture in an ultrasonic instrument for ultrasonic treatment for 30 min; 10000-;

2. The method of claim 1, wherein: the mass ratio of the soybean lecithin to the cholesterol is 12: 1.

3. The method of claim 1, wherein: the dosage of the phosphate buffer solution is 80 mL.

4. The method of claim 1, wherein: the rotational speed of the centrifugation is 20000 rpm.

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