Phospholipids—the Basic Composition of Liposomes

Phospholipids are the basic materials composing liposomes, which also determine the liposome’s physical and chemical properties. Factors affecting the stability of liposome formulations include the phase transition temperature and electric charges associated with phospholipids, the particle size associated with the manufacturing process, and the temperature, pH, and ionic strength associated with the environment. The type and amount of phospholipids are essential for the preparation of liposomes.

Classification of Phospholipids

Phospholipids are divided into phosphoglycerides and sphingomyelins by structure. Glycerol phosphate (PG) is composed of a hydrophilic polar head and two hydrophobic tails. The 1st and 2nd hydroxyl groups on the glycerol skeleton are esterified with fatty acids, and the 3rd hydroxyl group is esterified with phosphate groups linked to other groups. Phosphate linking groups are generally very small and are hydrophilic, and the most common types are choline, ethanolamine, serine, and inositol. Sphingomyelin (SM) is sphingosine, and there are amide bonds in the sphingomyelin molecules, which can form hydrogen bonding bands in the bimolecular membrane, which stabilizes the liposomes.

Phospholipids are divided into natural phospholipids and synthetic phospholipids by their sources. The natural phospholipids are mainly lecithin (phosphatidylcholine, PC), and the common types are soybean lecithin and egg yolk lecithin. The two fatty chains of PC of natural origin are different in length and saturation. Plant phospholipids have many unsaturated fat chains, which are liquid at normal temperature and are easily oxidized; animal phospholipids have many saturated fat chains, which are solid at room temperature and are not easily oxidized. Synthetic phospholipids are obtained by chemical synthesis reactions, mainly including DPPP (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine), etc., and they are generally saturated fatty chains with the characteristics of stability, high purity, strong oxidation resistance, product stability, etc., and thus is currently the preferred auxiliary material for the preparation of liposomes. Among them, lysophospholipids are substances with potential safety hazards that require focus. They are obtained by PC hydrolyzing one fat chain. As the name implies, due to the increased polarity, they have strong surface activity, and will insert into the erythrocyte membrane to dissociate them and cause hemolysis.

Physical Properties of Phospholipids

As the temperature rises, the acyl side chains in the phospholipid bilayer change from ordered to disordered arrangement, and the physical state of the lipid membrane changes from gel to liquid crystal, at the same time, the cross-sectional area of the membrane increases, the thickness of the bilayer decreases, the membrane fluidity increases, and the drug encapsulated in the liposome reaches the maximum release rate. During the process, the temperature at which this transition begins is called the phase transition temperature (Tm). The phase transition temperature of the liposome membrane is related to the length and unsaturation of the fat chain of the phospholipid molecule: the longer the fat chain, the higher the phase transition temperature; the more unsaturated bonds, the lower the phase transition temperature. In addition, Tm is also related to factors such as lipid composition in the membrane, encapsulated drugs, and ions in the aqueous phase, so this phase transition is more complicated than that of phospholipid. Differential scanning calorimetry (DSC) can be used to determine the phase transition temperature of lipids. When preparing liposomes, the temperature should be controlled to be higher than Tm, so that the drug enters the aqueous phase in the liposome. During normal storage, the temperature should be kept below Tm to reduce drug leakage.

Electric charge affects the liposome encapsulation rate and stability. Drug ions that are oppositely charged to the phospholipid membrane usually have a higher encapsulation rate, while drug ions of the same charge have a low encapsulation rate with a higher possibility to leak. The aggregation of liposomes is related to the Zeta potential. The higher the Zeta potential, the greater the electrostatic repulsion between the liposome particles, the less likely aggregation will occur, and the better the stability. Due to the presence of phosphate groups and/or amino groups, most phospholipids are neutral or negatively charged. Phosphatidylcholine (PC) and phospholipid ethanolamine (PE) are electrically neutral under physiological conditions. Phosphatidylserine (PS), phosphatidylinositol (PD), phosphatidylglycerol (PG) and diphosphatidylglycerol are all negatively charged phospholipids. Positively charged phospholipids are relatively rare, but there are positively charged lipids that modify liposomes, mainly including stearylamine (SA), cholesterol derivatives such as 3β-[N-(N’ N’-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate (DOTAP), etc.

In addition to the nature of phospholipids, the phase change temperature and charge of liposomes are also affected by ionic strength, pH, additives, and drugs. Therefore, the preparation of liposomes requires strictly control of the type and proportion of the composition to ensure the liposome’s uniform quality.

Chemical Properties of Phospholipids

The chemical stability of phospholipids directly affects liposome stability, such as oxidation and hydrolysis of phospholipids. Natural phospholipids are mixtures of various phospholipid molecules, most of which contain certain unsaturated fatty chains. Therefore, oxidized substances such as oxygen and oxidized free radicals are prone to oxidatively break unsaturated fatty chains to generate peroxides and malondialdehyde. Oxidative hydrolysis of phospholipids can occur during liposome preparation, storage, and application. Hydrolysis of phospholipids produces fatty acids and lysophospholipids, resulting in toxicity. Therefore, the preparations should be detected by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) to ensure safety (mainly lyso-phosphatidylcholine and lysophosphatidyl ethanolamine).

The physical and chemical properties of phospholipids determine the properties of liposomes. Therefore, it is necessary to add the oxidation index check item to the liposome quality control index. At the same time, due to safety concerns, the lyso-phospholipid check item should also be added to the quality study and stability inspection. In addition, in the prescription screening and process research, the phase transition temperature and charge of the bimolecular membrane should be studied. Only by adopting the above comprehensive measures, can the quality of liposomes be fundamentally controlled and the relevant information about the intrinsic decisive factors that affect the quality of liposomes be learned.