Comprehensive analysis, one article to understand the liposome encapsulation rate

PART 0 1.
What is liposome? In 1961, when Alec Douglas.
Bangham and RW Horne used negatively stained phospholipids to adjust the electron microscope, they observed under the electron microscope that the phospholipids formed a structure similar to the plasma membrane of a cell, and in 1964 they published an electron microscope photo taken by them
.
Further research found that when phospholipids are dispersed in water, multilamellar vesicles are formed, and each layer is a lipid bilayer, separated by water
.
They called this kind of artificial vesicles with a certain amount of water completely wrapped in a single or multi-layer concentric (or non-concentric) phospholipid bilayer as liposomes
.
Because the structure of liposome is similar to biological membrane, it is also called artificial biological membrane
.
The basic structure and type of liposomes can be divided into unilamellar liposomes and multilamellar liposomes
.
Vesicles containing unilamellar bilayer phospholipid membranes become unilamellar liposomes (ULV), or unilamellar liposomes
.
Unilamellar liposomes are divided into small unilamellar liposomes (SUV, particle size <100nm), namely nano liposomes, large unilamellar liposomes (LUV, particle size> 100nm) and huge unilamellar liposomes (GUV, particle size>1000nm)
.
Vesicles containing multi-layer bilayer phospholipid membranes are called multi-layer liposomes (MLV, particle size 100~1000nm)
.
Vesicles containing multiple unilamellar liposomes are called multilamellar liposomes (MVV, particle size> 1000nm)
.
PART 0 2.
What is the liposome encapsulation efficiency? Encapsulation efficiency is the key quality attribute of liposomes.
It refers to the percentage of the drug content encapsulated in the lipid bilayer to the total dose, which can reflect the degree of drug encapsulation in the liposome.
Guide the improvement of the preparation process
.
Due to the different properties of the encapsulated drugs and different liposome membrane materials, the best method to determine the encapsulation efficiency of each liposome often requires experimental investigation to determine
.
The key to the determination of the encapsulation rate is to separate the encapsulated drug from the unencapsulated free drug, and then use analysis methods such as spectroscopy and chromatography to detect the concentration of the encapsulated drug or free drug
.
Commonly used methods of encapsulation efficiency: centrifugation, ultrafiltration, centrifugation, dextran gel column, minicolumn centrifugation, dialysis and anti dialysis, protamine coacervation method, fluorescence method and the like
.
(1) Centrifugal method According to the different centrifugal speed, the centrifugal method is divided into low-speed centrifugation and ultra-centrifugation
.
The low-speed centrifugation method is suitable for fat-soluble drugs.
Its working principle is that the free fat-soluble drugs are insoluble in the aqueous medium required to dissolve liposomes, and are suspended in the system, using relatively small centrifugal strength and short centrifugation time , These undissolved free drugs will settle due to the centrifugal force, but the liposomes still exist in the supernatant to achieve separation
.
The ultracentrifugation method requires the centrifugal speed to be greater than 20 000 r/min, and the centrifugal time is generally longer than 30 min
.
During centrifugation, heat is generated by air friction, so a temperature-controlled centrifuge is required
.
In contrast to low-speed centrifugation, liposomes end up in the precipitate in ultra-centrifugation
.
Since liposomes and insoluble free drugs will settle together, the separation of the two cannot be achieved, so this method is suitable for the determination of drugs with better water solubility, which can ensure that the free drugs still exist in the supernatant
.
In addition, it should be noted that strong centrifugal force may cause particles to aggregate, destroy the bilayer structure of liposomes, and cause drug leakage
.
(2) Ultrafiltration centrifugation method The ultrafiltration centrifugation method is to put liposomes into an ultrafiltration tube equipped with an ultrafiltration membrane and centrifuge at an appropriate speed.
The free drug can pass through the ultrafiltration membrane under the action of centrifugal force.
Liposomes are trapped to achieve separation of the two
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Ultrafiltration centrifugation is mostly used to determine the encapsulation efficiency of water-soluble drug liposomes
.
However, the existence of the phenomenon of "concentration polarization" limits the application of ultrafiltration
.
Concentration polarization is due to the fact that in the ultrafiltration process, solvents and small molecular solutes can pass through the ultrafiltration membrane, while macromolecular solutes are trapped in the membrane, which will cause the concentration of macromolecular solutes on the surface of the ultrafiltration membrane to increase , Causing the osmotic pressure near the membrane to increase, preventing the solution from continuing to diffuse toward the ultrafiltration membrane, thereby reducing the membrane permeability of solvents and small molecules
.
(3) Dextran gel column method Dextran particles can form a gel-like structure after swelling, with a certain pore size inside, and small molecule free drugs can enter the pores to achieve a certain degree of retention; and lipids The body size is larger than the pore size of the gel, and liposomes cannot enter the pores, so a small amount of eluent can be eluted
.
The separation of liposomes and free drugs on the dextran gel column can be used to achieve the separation
.
(4) Micro-column centrifugation method Compared with the dextran gel column method, the volume of the eluate added by the micro-column centrifugation method is greatly reduced, which avoids the leakage of liposomes due to dilution
.
This method is to load the swollen dextran gel or pretreated ion exchange resin into a syringe, repeatedly equilibrate and centrifuge to form a dry microcolumn, and then add liposome suspension to the top of the syringe.
After a few minutes, add the eluent and set an appropriate centrifugal speed to elute the liposomes
.
If the gel has a strong adsorption effect on liposomes, this method cannot be used
.
In the experiment, attention should be paid to the choice of centrifugal rotation speed.
If the rotation speed is too large or too small, the column head of the gel column may break, and the free drug will also be eluted when the rotation speed is too high
.
(5) Dialysis and reverse dialysis method The dialysis method is to put liposomes into a dialysis bag that cuts off a certain molecular weight.
Generally, water or PBS buffer is used as the dialysis medium.
The free drug is transferred to the dialysis medium due to the difference in concentration inside and outside the dialysis bag.
, And liposomes are trapped in the dialysis bag because of their larger particle size, so the two are separated
.
However, fat-soluble free drugs have poor solubility in the dialysis medium and will accumulate on the surface of the dialysis membrane, block the micropores on the membrane and cannot enter the dialysis fluid.

.
In order to meet the drain condition in the dialysis test, a large amount of dialysis medium is needed, which will undoubtedly dilute the entire liposome system, destroy the dynamic balance between the liposome and the surrounding free drug, and even cause the leakage of the liposome, and longer dialysis Time also has high requirements on the stability of liposomes
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The anti-dialysis method can avoid the above problems
.
It puts liposomes outside the dialysis bag, and the dialysis bag is filled with dialysis medium
.
Since the amount of dialysis medium is greatly reduced, the leakage of liposomes due to dilution can be effectively avoided
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(6) Other methods Protamine agglomeration method: Protamine is a basic protein, which is positively charged.
It can combine with negatively charged or neutral liposomes to form a polymer.
The density increases.
After centrifugation, the fat The plastid-protamine polymer is precipitated and separated from the free drug
.
Solid phase extraction: Using the principle of chromatographic adsorption, free drugs are adsorbed on the SPE column stationary phase with similar polarity, while the liposomes are not retained on the SPE column and can be eluted with a small amount of water
.
The solid-phase extraction method has a higher degree of separation between liposomes and free drugs, because it uses more characteristic and more stable adsorption capacity to achieve separation
.
However, the method is more complicated.
The strength of the adsorption between the drug and the SPE column stationary phase is affected by many factors, and a lot of experiments are needed to find the best experimental conditions
.
Fluorescence method: Most of the currently used methods for measuring the encapsulation efficiency require the separation of liposomes and free drugs first, while the fluorescence method does not require separation, as long as the different fluorescence characteristics of encapsulated drugs and free drugs are used to compare liposomes.
The change of fluorescence intensity before and after demulsification can calculate the encapsulation efficiency
.
Table 1 Comparison of several methods for determining the encapsulation efficiency of drug-loaded liposomes.
Remarks: EPC: egg yolk lecithin; CHOL: cholesterol; DPPC: dipalmitoylphosphatidylcholine; DCP: sphingomyelin; GMS: mono- and diglycerin fatty acids Ester; SPC: soybean lecithin; DDAB: didodecyl dimethyl ammonium bromide; PEG2000: polyethylene glycol 2000; DSPE-PEG2000: distearoyl phosphatidylethanolamine-polyethylene glycol 2000; PC: Lecithin; DSPE-PEG2000-NHS: Distearoylphosphatidylethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide; DOTAP: (2,3-dioleoyl-propyl)-trimethylamine; HSPC :Hydrogenated soybean lecithin;DOPE:Dioleoylphosphatidylethanolamine PART 0 3.
How to choose the most suitable method for measuring the encapsulation efficiency? Low-speed centrifugation is suitable for fat-soluble drugs.
It is simple to operate and not easy to destroy the liposome membrane structure, but liposomes and free drugs are often not completely separated; ultracentrifugation is mostly used for water-soluble drugs and requires liposome membrane structure A certain degree of hardness can withstand the influence of high speed
.
The ultrafiltration centrifugation method has a wide range of applications.
There are many types of ultrafiltration membrane materials and molecular weight cutoffs, which can meet the multiple needs of the experimenter; its disadvantage is that concentration polarization may occur, which may dilute the liposomes to a certain extent The solution to be tested can avoid this phenomenon
.
Both the dextran gel column method and the microcolumn centrifugation method use the principle of molecular exclusion for separation, but if the drug is not retained on the gel column or is too strongly adsorbed, these two methods cannot be used
.
The dextran gel column method dilutes the liposome system due to the addition of a large amount of eluent, which may cause liposome leakage
.
However, the column volume of the microcolumn centrifugation method is small, and the volume of the added eluent is also small, which will not cause liposome leakage, but it is necessary to check the centrifugal speed to obtain the best separation effect
.
The dialysis method is also a common method to determine the encapsulation efficiency, which is suitable for water-soluble drugs, but the dialysis time is generally longer than 36 hours, which has high requirements for liposome stability; in addition, the addition of a large amount of dialysis medium will also dilute the liposomes System, may also cause liposome leakage
.
The reverse dialysis method greatly reduces the amount of dialysis medium, and better avoids the leakage of liposomes due to dilution
.
The protamine aggregation method is only suitable for negatively charged and neutral liposomes.
Although the solid phase extraction method has good results in separating liposomes and free drugs, the experimental method is complicated and it is difficult to find the best separation conditions.

.
Fluorescence method can be used to measure the encapsulation efficiency in the coexistence of liposomes and free drugs.
It does not require separation of the two, but generally requires the introduction of a fluorescent indicator, and a certain fluorescence reaction occurs to calculate the encapsulation efficiency
.
According to the nature of the analyte and the characteristics of various determination methods, the decision tree for the determination method of liposome encapsulation efficiency is summarized, as shown in Figure 1
.
Figure 1 Decision tree for the selection of liposome encapsulation efficiency determination method PART 0 4.
What are the factors that affect the liposome encapsulation efficiency? There are many factors that affect the encapsulation efficiency of liposomes, including the composition of the lipid membrane, phospholipid chain length, charge, particle size, preparation method, and drug properties.
Choose one or more of them to improve it.
Improve or increase the encapsulation efficiency of liposomes
.
Lipid membrane composition and liposome potential The composition of the lipid bilayer membrane affects the distribution and encapsulation efficiency of the drug in the membrane
.
Liposomes are prepared from phosphatidylcholine (PC) or its hydrogenated derivatives extracted from soybean or egg yolk
.
Natural phospholipids contain unsaturated fatty acyl chains, and they are easily oxidized and decomposed, which affects the stability of liposomes
.
Drug properties Factors such as the solubility, polarity and matching degree of the bilayer of the drug itself will affect the encapsulation efficiency
.
Drugs with high lipophilicity or high hydrophilicity, that is, drugs with lgPoct between 4.
5 and -0.
3, can form liposomes with higher encapsulation efficiency
.
Therefore, it is possible to consider salting or esterifying the drug to form liposomes to improve the encapsulation efficiency or stability of liposomes
.
PART 0 5.
How to calculate the encapsulation rate? "Chinese Pharmacopoeia" 2005 edition (Part 2) appendix "Guidelines for Microcapsules, Microspheres and Liposome Preparations" under the quality inspection items: If the obtained is microcapsules, microspheres, lipids dispersed in a liquid medium The body should be separated and determined by an appropriate method (such as gel chromatography, centrifugation or dialysis), and the encapsulation efficiency should be calculated by the following formula
.
Encapsulation rate (%) = the amount of encapsulated medicine in the system/the total amount of encapsulated and unencapsulated medicine in the system × 100% The encapsulation rate shall not be less than 80%
.
PART 0 6.
Outlook The existing methods for determining the encapsulation efficiency basically use the difference in particle size between liposomes and free drugs to separate the two first, and then accurately determine the concentration of one side
.
However, this physical difference is not strong and unstable, and it is susceptible to influence and interference
.
In the future, methods for measuring the encapsulation efficiency should be more combined with spectroscopy and chromatography to increase the specificity and reliability of the method.
It can also take advantage of other differences in physical and chemical properties between liposomes and free drugs to develop New methods with different working principles, and various methods can mutually confirm each other, making the measurement results more reliable
.