*3.2. Double Emulsion*

Liposomes can be also prepared through the Double Emulsion technique which involves lipids dissolution in a water/organic solvent mixture. The organic solution, containing water droplets, is mixed with an excess of aqueous medium, leading to a water-in-oil-in-water (W/O/W) double emulsion formation. After mechanical vigorous shaking, part of the water droplets collapse, giving Large Unilamellar Vesicles (LUVs) [76]. This method was used for NABDs encapsulation, but achieved a very low entrapment efficiency [77].

## *3.3. Reverse Phase Evaporation*

The Reverse Phase Evaporation (REV) method also allows one to achieve LUVs loaded with NABDs [78]. In this technique a two-phase system is formed by phospholipids dissolution in organic solvents and aqueous buffer. The resulting suspension is then sonicated briefly until the mixture becomes a clear one-phase dispersion. The liposome formation is achieved after the organic solvent evaporation under reduced pressure. This technique has been used to encapsulate different large and small hydrophilic molecules included nucleic acids, i.e., in a work of Stuart and Allen, cationic vesicles loaded with nucleic acids and covered with neutral lipids, with significantly high incorporation efficiency (80–100%), and being stable in 50% human plasma at 37 ◦C, were produced [79]. Recently by means of a modified REV, Mokhtarieh and collaborators have produced Asymmetric Liposome Particles (ALPs) containing siRNA of about 200 nm in size with more than 90% encapsulation efficiency. The method provides the formation of two kinds of lipid inverted micelles composing the inner and outer lipid film. The siRNAs are entrapped in the inner one, made of ionizable cationic lipids, which is mixed with the outer lipid film made of conventional lipids. After a solvent evaporation and dialysis, siRNA-ALPs are achieved [80].

#### *3.4. Microfluidic Method*

A relatively new technology used for liposomes production is the Microfluidic method, unlike other bulk techniques, this one gives the possibility of controlling the lipid hydration process. The method can be classified in continuous-flow microfluidic and droplet-based microfluidic, according to the way in which the flow is manipulated [81]. In 2004, Jahn and collaborators described a microfluidic hydrodynamic focusing (MHF) method which operates in a continuous flow mode. Briefly lipids are dissolved in isopropyl alcohol which is hydrodynamically focused in a microchannel cross junction between two aqueous buffer streams. Vesicles size can be controlled by modulating the flow rates, thus controlling the lipids solution/buffer dilution process [82]. The method was successfully extended for producing oligonucleotides (ON) lipopolyplexes by using a microfluidic device consisting of three-inlet and one-outlet ports. Lipids ethanol solution and ON aqueous solutions are contained into sterile syringes connected to the inlet ports. Briefly, a fluid stream is split into two side streams at inlet port 1 or 2, while a fluid stream directly entered the center microchannel through inlet port 3. The resulting ON liposomes (about 115 nm in size) solution is collected at the outlet port [81].

#### *3.5. Dual Asymmetric Centrifugation*

Dual Asymmetric Centrifugation (DAC) is another method for the production of NABDs-liposomes [83]. This technique differs from the usual centrifugation because the sample is undergone, during the normal centrifugation process, to an additional rotation around its own vertical axis. By this way an efficient homogenization is achieved due to the two overlaying movements generated: The sample is pushed outwards, as in a normal centrifuge, and then it is pushed towards the center of the vial due to the additional rotation. Briefly, by mixing lipids and an NaCl-solution a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain liposomal dispersion. Liposome size can be regulated by optimizing DAC speed, lipid concentration and homogenization time. With this method Hirsch and coworkers have prepared siRNA-liposomes, about 109 nm in size, with high entrapping efficiency, ranging from 43 to 81%, depending upon batch size [84]. In 2011, also Adrian and collaborators have used the DAC method to produce liposomes containing siRNA, targeting the particles surface with an antibody for the selective interaction with neuroblastoma cells, achieving 190 to 240 nm particles with siRNA encapsulation efficiency of up to 50% [85]. By this technique, in only one step, sterile SUVs formulations in a highly reproducible manner can be prepared [86].
