**4.2 Microfluidic systems for electrostatic complexation of DNA**

The electrostatic interactions between DNA and cationic liposomes produce particles with different sizes and morphology (Mannisto et al., 2002; Oberle et al., 2000) that depend on R+/-, buffer ionic strength, order of component addition, reaction conditions, and the type of lipids (Mount et al., 2003; Zelphati et al., 1998). In this context, HF can also be used to control the diffusion process for DNA compaction, producing well organized aggregates. The flow velocity is the major parameter controlling the aggregation process (Dootz et al., 2006).

Otten et al. (2005) investigated the HF microfluidic device to produce cationic liposome-DNA complexes. The DNA solution is introduced in the central stream and the cationic liposome stream introduced at a lateral position. The average liposome size was 200 nm (composed of 1:1 DOTAP and DOPC with a lipid concentration of 25 mg.mL-1), and the DNA was calf thymus (5 mg.mL-1). The flow velocity was 100 mm.s-1, varying with vLiposome = 13vDNA and vLipossomas = 130vDNA, where v is the flow velocity. The authors concluded that the complex is formed in two steps. The first step relates to the formation of a multilamellar complex, followed by the second step in which DNA is organized inside the lamellae. The central stream can be focused according the FRR, and reducing the diffusional length allows faster mixing (Knight et al., 1998).

Technological Aspects of Scalable Processes for the

**scalable top-down processing** 

**6.1.1 Statistical analysis** 

respectively.

before characterization.

Production of Functional Liposomes for Gene Therapy 281

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

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

**6. A case study of the production of cationic liposomes and gene vaccines in** 

Although top-down strategies are important and high shear processing has various uses, no systematic studies have been carried out on the effects of liposome comminution on the physico-chemical and surface properties of liposomes. The data in the literature are sparse

Aiming to produce the gene vaccine composed of EPC/DOTAP/DOPE liposomes with DNA complexed on their external surface, we initially studied the significance of the process variables for the properties of liposomes composed of egg lecithin. Mechanical forces were used for homogenation and comminution in Caules type stirrer, Ultra-Turrax, and microchannel microfluidizer equipment. A main variable was selected and its effects on the physico-chemical properties of the liposomes characterized. Finally, a scalable discontinuous process was established and EPC/DOTAP/DOPE liposomes produced and complexed with DNA. The physico-chemical and biological properties of the gene vaccine

controlled by the relative flow rates between the phases and by microchannel length.

in regards to the kind of impellers, comminution equipment, or type of lipids used.

were compared with our previous gene vaccine prepared using Bangham's method.

We studied the effects of the shear rate using multi-factorial statistical experimental planning (Montgomery 2008) in order to delineate the relative importance and influence of the shear rate and feed flow rate on the mean diameter, polydispersity, zeta potential, and viscosity of egg lecithin liposomes. The liposomes were prepared with high lipid concentration (300 mM), aiming for applications in scaling up processes. Food grade egg lecithin (60% phosphatidylcholine content) from Degussa (GmbH Germany) and ethanol 99- 100% from Labsynth ltda. (São Paulo- Brazil) were used as the lipid and solvent,

Figure 6 shows the experimental outline for liposome preparation. The 300 mM ethanollipid suspension in a beacker (1) was fed at a previously defined flow rate through a peristaltic pump (2) into the bottom of a 150 mL beaker with four fins containing pure Milli Q water (3). Continuous mechanical stirring was provided by a Caules type stirrer or Ultra-Turrax® IKA T25 (Ika Works) (4). The final lipid concentration was 50 mM. After the feeding was complete, stirring was maintained for an additional 15 min. Comminution was also carried out in a microchannel microfluidizer (Microfluidizer® M-110P) with 100 mL of a liposome dispersion pre-processed through an Ultra-Turrax® at 5000 to 21000 s-1 shear rate and 0.09 to 0.96 mL.s-1. The microfluidizer worked in the pressure range of 200 to 1500 bar and for various passages. All liposome dispersions were stored at 8°C for 12 hours

**6.1 Effects of the process variables on liposome properties** 

consumption and the elimination of organic solvents, is still a challenge.

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 aggregation process between DNA and cationic liposomes.
