**5. Physico-chemical aspects**

The processes that use top-down strategies for liposome production differ according to the remotion of solvent and/or application of shear. The remotion of solvent is carried out by evaporation (Bangham´s method or multitubular system), spray drying or evaporation in reverse phase, sublimation (dehydration–hydration), or solubilization in water by ether/ethanol injection in diluted or concentrated phases. The incomplete remotion of solvent has a direct impact on liposome size.

We have observed that the presence of organic solvent in spray-dried lipid structures generates more amorphous or more crystalline domains due to the packing of lipids in the bilayers (Alves and Santana, 2004). The operational conditions interfere with the drying rate, and mass transfer limitations result in the complete evaporation of ethanol. The higher the evaporation rate, the higher the amount of ethanol remaining inside the particles because a shield of packed lipids close to the surface and more amorphous structures are formed. At a slower rate of evaporation, it is more controlled, generating more crystalline structures. The subsequent hydration of the solid particles with different levels of crystallinity influences the size and polydispersity of the generated liposomes.

Similar mass transfer limitations are present when the organic solvent is removed by diffusion in water. A compromise between the diffusion and vesiculation rates controls particle size. The opposite rates of diffusion and hydration depend on the barrier created by the phospholipids, which is a function of its concentration and depends on the interaction among lipids. In previous studies, we characterized the packing of pseudo-ternary mixed Langmuir monolayers of EPC/DOTAP/DOPE using surface–molecular area curves. The interactions and miscibility behavior were inferred from the curves by calculating the excess free energy of the mixture (ΔGExc). The deviation from ideal showed dependence on the lipid polar head type and monolayer composition (Rigoletto et al., 2011).

The rates of vesiculation and solvent diffusion are comparable in magnitude only at very low lipid concentrations, generating small liposomes in an excess of water. Under other conditions, the barrier created by the extension of the primary aggregation of lipids delays solvent remotion, generating large multilamellar liposomes. Otherwise, the rate of vesiculation is associated with the hydrophobicity of the non-polar groups. Therefore, the vesiculation rate in the primary aggregate controls liposome size. When cationic lipids are used, such as in the DOTAP/DOPE/EPC system, the electrostatic repulsion among the molecules benefits solvent diffusion and hydration, generating smaller liposomes.

Because of the softness of the primary aggregate and the bilayer fluidity imparted by the phase transition temperature, liposomes are prone to fusion due to the non-ordered Brownian movement of the colloids in the medium, and multilamellar vesicles are generated in a broad range of sizes. The remaining solvent generates polydispersed liposomes, which are more favorable for fusion. However, charged lipids stabilize liposomes. Therefore, mass transfer is critical in discontinuous processing, controlling liposome size and polydispersity. Despite limitations in mass transfer and interactions, discontinuous processes are still preferable due to the simplicity in carrying out massive liposome production and scaling up

A micromixer (Jellema et al., 2010) and multi-inlet microfluidic HF (MF) system (Koh et al., 2010) were recently investigated as alternatives for producing the liposome-DNA complexes. These studies point out new and promising alternatives aiming to control the

The processes that use top-down strategies for liposome production differ according to the remotion of solvent and/or application of shear. The remotion of solvent is carried out by evaporation (Bangham´s method or multitubular system), spray drying or evaporation in reverse phase, sublimation (dehydration–hydration), or solubilization in water by ether/ethanol injection in diluted or concentrated phases. The incomplete remotion of

We have observed that the presence of organic solvent in spray-dried lipid structures generates more amorphous or more crystalline domains due to the packing of lipids in the bilayers (Alves and Santana, 2004). The operational conditions interfere with the drying rate, and mass transfer limitations result in the complete evaporation of ethanol. The higher the evaporation rate, the higher the amount of ethanol remaining inside the particles because a shield of packed lipids close to the surface and more amorphous structures are formed. At a slower rate of evaporation, it is more controlled, generating more crystalline structures. The subsequent hydration of the solid particles with different levels of crystallinity influences

Similar mass transfer limitations are present when the organic solvent is removed by diffusion in water. A compromise between the diffusion and vesiculation rates controls particle size. The opposite rates of diffusion and hydration depend on the barrier created by the phospholipids, which is a function of its concentration and depends on the interaction among lipids. In previous studies, we characterized the packing of pseudo-ternary mixed Langmuir monolayers of EPC/DOTAP/DOPE using surface–molecular area curves. The interactions and miscibility behavior were inferred from the curves by calculating the excess free energy of the mixture (ΔGExc). The deviation from ideal showed dependence on the

The rates of vesiculation and solvent diffusion are comparable in magnitude only at very low lipid concentrations, generating small liposomes in an excess of water. Under other conditions, the barrier created by the extension of the primary aggregation of lipids delays solvent remotion, generating large multilamellar liposomes. Otherwise, the rate of vesiculation is associated with the hydrophobicity of the non-polar groups. Therefore, the vesiculation rate in the primary aggregate controls liposome size. When cationic lipids are used, such as in the DOTAP/DOPE/EPC system, the electrostatic repulsion among the

Because of the softness of the primary aggregate and the bilayer fluidity imparted by the phase transition temperature, liposomes are prone to fusion due to the non-ordered Brownian movement of the colloids in the medium, and multilamellar vesicles are generated in a broad range of sizes. The remaining solvent generates polydispersed liposomes, which are more favorable for fusion. However, charged lipids stabilize liposomes. Therefore, mass transfer is critical in discontinuous processing, controlling liposome size and polydispersity. Despite limitations in mass transfer and interactions, discontinuous processes are still preferable due to the simplicity in carrying out massive liposome production and scaling up

molecules benefits solvent diffusion and hydration, generating smaller liposomes.

aggregation process between DNA and cationic liposomes.

**5. Physico-chemical aspects** 

solvent has a direct impact on liposome size.

the size and polydispersity of the generated liposomes.

lipid polar head type and monolayer composition (Rigoletto et al., 2011).

processes. In this context, the development of technologies that promote the production of cationic liposomes with controlled size and low polydispersity index, with low energy consumption and the elimination of organic solvents, is still a challenge.

Continuous processes in microchannels with HF reduce the limitations due to mass transfer in the interface of the primary aggregate, and the continuous operation decreases interactions among particles due to Brownian movement. Therefore, particle size is controlled by the relative flow rates between the phases and by microchannel length.
