*4.6.3 Preparation method (incorporating drugs)*

In addition to the ability to entrap drugs with different solubility characteristics, it has been hypothesized that liposomes have different release kinetics. In general, multilamellar liposomes are more easily formed at larger hydrodynamic diameters, and, therefore, they have greater entrapped volumes than unilamellar liposomes. As a result of this, unilamellar liposomes with a hydrodynamic diameter of 130 nm exhibit a much faster release rate than multilamellar liposomes with two to three lamellar bilayers and a hydrodynamic diameter of 250 nm [123, 124]. The difference in the release rate is overall due to the number of phospholipid bilayers that it has to cross before being released [123, 124]. The ongoing interest of researchers in liposomal characteristics such as their stability, pharmacokinetic properties, and therapeutic efficacy has led to second-generation liposomes through the modulation of their lipid composition, size, and the charge of the vesicle. The addition of cholesterol to the lipid bilayer of liposomes reduces their permeability, increases their in vivo and in vitro stability, and can be used to anchor other molecules such as polyethylene glycol (PEG) or deoxyribonucleic acid (DNA) to the liposomes for their application in biosensing or as "stealth" drug carriers [124]. The use of phosphatidylcholine with saturated fatty acyl chains and materials that stretch transition temperatures beyond 37°C offered even greater stabilization [115, 125]. Furthermore, hydrophilic carbohydrates or polymers, such as monosialoganglioside (GM1) and PEG, were included in the liposomal composition. GM1 can lead to the prolongation of the in vivo liposome viability time [126–129].

There are many different methods to prepare liposomes. According to Bozzuto and Molinari [130], the choice of the method depends on factors such as the physicochemical characteristics of liposomes and/or drug components; the toxicity and concentration of the loaded drug; the type of medium in which liposomes are dispersed; the additional process during the application/delivery of liposomes; the desired size for the application; the half-life desired for successful application; costs, reproducibility, and applicability regarding large-scale production for clinical purpose; and good manufacturing practice-relevant issues. In addition, the target organ is a significant issue to be considered when planning the preparation of liposomes.

Every method for preparing liposomes involves four basic stages: drying down lipids from an organic solvent, dispersing the lipid in aqueous media, purifying the resultant liposome, and analyzing the final product. The most common methods for producing liposomes, according to Gabizon et al. [123, 128] and Akbarzadeh et al., [131] are the following.
