**3. Liposomes to LNPs**

Liposomes are the progenitors of all LNPs, and they are formulated directly from lipid constituents (Figure 3). Liposomes can be prepared by various techniques. The method based on hydration of lipid film represents the most exploited technique for preparation of lipo‐ somes. The lipids of known molarities are dissolved in organic solution which is subsequently slowly evaporated in vacuo to produce a thin film. The film is hydrated with a suitable aqueous buffer at temperature, which is generally above transition temperature of lipids used. Aqueous buffer contains also compound which are to be entrapped into liposomes. Size of liposomes can be reduced by freeze-thaw procedure followed by sonication, extrusion, microfluidisation or high pressure homogenisation. These procedures can tune size distribution of final liposo‐ mal preparation to desired values. The main parameters characterising liposomes are their size, spherical shape and degree of lamellarity. Liposomes may consist of one bilayer (unila‐ mellar), several bilayers (oligolamellar) or multiple bilayers (multilamellar). Membrane rigidity can be adjusted using appropriate lipids and membrane fluidity may be controlled with the use of phospholipids with higher or lower transition temperature.

**Figure 3.** Various morphological types of liposomes and real appearance in cryoelectron microscopy.

Small unilamellar vesicles (SUV); Large unilamellar vesicles (LUV); Multilamellar vesicles – concentric (MLV); Multivesicular vesicles (MVV)

Liposomes are generally formulated on the nm scale and can be further size refined by passage through physical membrane pores of known size (extrusion). Liposomes are typically charac‐ terised by their size, spherical shape and degree of lamellarity. They may be composed of a single bilayer (unilamellar), a few bilayers (oligolamellar), or multiple bilayers (multilamellar). The rigidity of the membrane can also be modified with the use of suitable lipids; and the fluidity of the membrane may be controlled using phospholipids with higher or lower LαI − HII mesophase transition temperatures. In general lipids of stearic acids (fully saturated C18 hydrocarbon chains) bestow rigidity to liposome structures (by favouring LαI fluid mesophas‐ es), whilst lipids of oleic acid (Δ9 unsaturated C18 hydrophobic chains) can result in less stable structures (by favouring HII fluid mesophases). Liposomes have traditionally been used as some of the very earliest nanodrug delivery vehicles, encapsulating water-soluble drugs within their central aqueous cavities in order to improve drug pharmacokinetics [6-8]. Liposome spherical diameters can be investigated by using scattered light off the surface of the particles with photon correlation spectroscopy. Alternatively cryo-TEM can be used to provide visual images of liposome sizes and shape. The surface charge of liposomes can be obtained by measuring their zeta potential (electrokinetic potential). Liposome associated techniques and characteristics have all been employed in more recent time times to prepare more chemically diverse LNPs.
