**4. Technological processes**

Most of the conventional methods for liposome production require an additional unit operation for size reduction and polydispersity, as they are top-down approaches. In this approach, liposomes are produced from the hydration of a thin film of lipids using Bangham's method (Bangham et al., 1965), multitubular system (Torre et al., 2007; Tournier et al., 1999), detergent depletion or emulsion methods, ether/ethanol injection, and reverse phase evaporation (Lasic, 1993; New, 1990). All of these processes are discontinuous and only the ether/ethanol injection and multitubular system are scalable. Shearing or impact strategies are

Technological Aspects of Scalable Processes for the

A B

acquisition (adapted from Zhang et al., 2008).

al., 2010).

Production of Functional Liposomes for Gene Therapy 277

to lipid/solvent flow) correspond to smaller liposome diameter (Janh et al., 2010). However, caution is required in this flow rate analysis as the final alcohol content in the liposome colloidal dispersion can change with FRR and as a consequence of particle size. Alcohol can be used to disrupt liposomes and, at low concentrations, liposome size can be increased due to alcohol incorporation into the bilayer. Precise analysis can be performed if the alcohol is removed after liposome processing. By decreasing the total volumetric flow rate, the residence time can be increased and a lower average vesicle diameter and narrower size distribution obtained. This behavior indicates that if increasing the total volumetric flow rate, the microchannel length must be longer to complete the alcohol diffusion, otherwise large particles will be obtained due to bulk mixing downstream of the channel (Janh et

Fig. 4. (A) Schematic representation of hydrodynamic focusing (HF) in a microfluidic device with four-channel intersection geometry. The organic solvent (miscible in water) containing the dispersed lipids is injected in the middle stream and hydrodynamically compressed by two aqueous (or buffered) streams. The flow is in the x direction and alcohol diffusion is in the y direction. (B) Schematic representation of the experimental apparatus for liposome production using HF: (1) microfluidic device; (2) water syringe pumps; (3) syringe pump for

The lipid concentration in alcohol is also another important parameter for controlling size. The development of vaccines for *in vivo* applications requires the highest drug-loading capacity. In terms of gene vaccines, the DNA-loading capacity correlates with the cationic lipid content, defined by the molar charge rate (R+/-) between the cationic charges (from the cationic lipid) and negative charges (from the DNA). The R+/- and total lipid content are project parameters for scaling up (or scaling out) processes. As an example, the R+/- of the tuberculosis gene vaccine is 10 and the total lipid concentration 64 mM (Rosada et al., 2008). These parameters require cationic liposome production in a high lipid concentration under unusual microfluidic conditions. Aiming to explore the effect of lipid concentration on microfluidic HF processes, we investigated the influence of high lipid content (100 mM) and lipid composition in ethanol (EPC or EPC/DOTAP/DOPE) as a function of average size. We understand that the studied molar concentration is greater than the lipid solubility in ethanol (approximately 4 mM for EPC) (New, 1990) and, in this case, the lipid dispersion offers an additional barrier to ethanol diffusion into water. Based on this assumption, we simulated the required microchannel length for complete ethanol diffusion from a central stream (after HF) as a function of the lipid concentration in the ethanol stream considering

lipids/ethanol stream; (4) collector flask; (5) stereo microscopy; (6) computer data

the general key for homogenation and reducing liposome size. Mechanical stirring, extrusion through orifices (French press), extrusion through membranes, high-pressure impactor, and microchannel microfluidizer are equipment used in high-energy processes.

Bottom-up processes are low energy processes inspired by biological systems and used for the development of functional nanomaterials, such as supramolecular structures, selfaggregated monolayers, Langmuir-Blodgett films, aggregated peptide nanotubes, and deposited polyelectrolites or proteins in multilayers (Mijatovic et al., 2005; Shimomura & Sawadaishi, 2001).

Microfluidic systems have been the main representatives of bottom-up processing for liposome production in continuous and scaled up processes. Different microfluidic systems can be applied to the production of liposomes and giant liposomes (Ota et al., 2009; Shum et al., 2008; Wagner et al., 2002). DNA complexation on the external surface of liposomes also constitute a promising technology for the production of gene vaccines.
