Quality Control Of Liposomes 2
As a promising drug carrier, liposomes require strict quality control for clinical use. In the previous article, we have introduced the requirements and methods of liposome morphology and particle size control. This article will introduce other parameters involved in liposomes as drug carriers，including: encapsulation rate, oxidation index, lysophospholipid, drug content, leakage rate, osmotic pressure and sterilization conditions.
Encapsulation efficiency is an important indicator to measure the quality of liposomes. It refers to the ratio of the encapsulated drug to the total amount of drug in the liposome suspension. The key to determining the encapsulation efficiency is to separate the unencapsulated free drug from the liposome suspension without destroying the liposome. Commonly used separation methods include gel column permeation chromatography, dialysis, ultracentrifugation and so on.
- Gel Column Permeation Chromatography
Dextran gel and agarose gel are commonly used column chromatography packing, and their separation principle is equivalent to molecular sieve. When the liposome suspension is eluted by the eluent, the free drug enters the small pores inside the gel particles, and the liposomes are eluted from the column through the gaps between the gel particles. In the elution sequence, the drug-loaded liposomes are eluted first, and the free drug flows out of the column because of the longer elution “path”.
- Dialysis
The small holes on the dialysis bag can trap large molecules and let small molecules pass through, which can effectively separate drug molecules (except macromolecular drugs) and liposomes. The selection of dialysis bag specifications is similar to that of gel. The molecular weight cut-off is for linear molecules such as proteins. For a drug molecule with a smaller molecular weight, the molecular space volume is larger, so you need to choose a dialysis bag with a larger molecular weight cut-off. The recovery rate of free drug is usually selected as an indicator to ensure that drug molecules can smoothly pass through the dialysis bag without leakage of liposomes. The dialysate medium needs to have high drug solubility and cannot destroy the liposome structure.
Oxidation Index
Phospholipids are easily oxidized, which is a prominent problem in liposome preparation and quality control. The oxidation of phospholipids is divided into three stages: single double bond coupling, oxidation product formation, acetaldehyde formation and bond breakage. Because the products of each stage are different, the degree of oxidation is difficult to evaluate with an experimental method. The oxidation index is usually used as the criterion. The phospholipid after oxidative coupling has an ultraviolet absorption peak around 230nm, which is different from the unoxidized phospholipid. Therefore, the absorbance of liposomes at wavelengths of 233nm and 215nm after being dissolved in absolute ethanol were measured respectively, and the oxidation index is the ratio of the two. Generally, the oxidation index should be controlled below 0.2.
Lysophospholipid
Liposomes prepared with unsaturated fatty chain phospholipids, under certain conditions, the unsaturated double bonds of phospholipids are prone to oxidation and cleavage to form lysophospholipid, which makes liposomes have a certain safety hazard when injected and administered. So it is necessary to check the product The lysophospholipid is checked, and the lysophospholipid is generally controlled not more than 3%. Thin-layer chromatography (TLC) can be used to determine lysolecithin.
Drug Content
Liposome components such as phospholipids and cholesterol have absorption in a wide wavelength range in the ultraviolet region, which may overlap with the ultraviolet absorption of conventional drugs. Therefore, it is generally not possible to directly measure the drug content by ultraviolet spectrophotometry. It is recommended to use the highly specific HPLC method for drug content analysis, and do a good job of system suitability test to ensure that the drug and excipients have good separation. The content determination method is verified through specificity test, linearity test, recovery test, precision test, repeat test and stability test.
The recovery rate verification of the drug content determination is consistent with other common preparations. Mix the main medicine with the high, medium, and low proportions of excipients, and focus on whether the medicine is completely extracted in the presence of the excipients and whether the excipients interfere with the drug determination. The excipients used can be directly mixed with the drugs, or the excipients can be made into blank liposomes and then mixed with the drugs. During the determination process, it is still necessary to pay attention to whether the liposome destroyer can completely destroy the liposome and whether the drug is completely released.
Leakage Rate
There are generally three types of drugs in liposomes, namely adsorption, encapsulation and embedding. The drug, liposome, and surrounding solution are in a state of equilibrium. The drug in the liposome suspension is affected by the concentration gradient of the solution or the plasma protein binding and other factors, causing drug leakage. Therefore, it is necessary to test the stability of the liposome in the in vitro PBS solution or the plasma protein solution, that is, the leakage rate. In actual operation, liposome preparations are usually diluted 10-fold with PBS buffer or plasma protein solution. The measurement method is the same as the determination of the encapsulation rate, and the leakage rate curve (the ordinate is the leakage rate and the abscissa is the time) is used for evaluation.
Osmotic Pressure
The liposome preparations currently developed are mostly injections. The osmotic pressure of human blood ranges from 285 to 310 mOsm. In order to achieve isotonicity, the diluent or aqueous phase outside the liposome is usually normal saline.
Sterilization Conditions
The general methods of sterilization include filtration sterilization, aseptic operation, radiation irradiation (60Co irradiation, 15-20kGy), 121C autoclaving and so on. Because the liposome phase transition temperature is low, the use of autoclaving can easily cause changes in the liposome bilayer membrane and cause drug leakage. Therefore, autoclaving is generally not suitable. The common sterilization methods of liposomes are filtration sterilization and aseptic operation. The higher standard sterilization technology increases the difficulty of large-scale liposome production.