**1. Introduction**

266 Non-Viral Gene Therapy

Zanta, M. A., Beleguise-Valladier, P. & Behr, J.-P. (1999) Gene delivery: A single nuclear

*Acad. Sci. U.S.A.* 96, 91–96.

localization signal peptide is sufficient to carry DNA to the cell nucleus. *Proc. Natl.* 

The success of gene delivery systems in *in vivo* or *in vitro* applications depends on efficient transfection. Cationic liposomes remain a promising alternative for nonviral DNA carriers, mainly because they protect DNA from interstitial fluids and easily interact with cells (Gregoriadis, 1993; Lasic, 1997). However, in order to be effective in the immunological response, cationic liposomes must be functional and reach their specific target. Stability, reduced toxicity, efficiency in delivering genes to cells, and specific targeting to the nucleus are essential requirements for prophylactic and/or therapeutic performance. In order to achieve these standards, important physico-chemical parameters in liposomes must be controlled, such as the functionality of the lipids, the concentration of the cationic lipid, DNA loading (reflected by the R+/- molar charge ratio), the zeta potential, size, and polydispersity.

Several laboratory experiments have already explored DNA vaccines using cationic lipids. A classical investigation of lipid functionality and composition, as well as the efficiency of cationic liposomes as DNA carriers, was performed by Perrie and colleagues (Perrie & Gregoriadis, 2000; Perrie et al., 2001). The plasmid pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was encapsulated in dehydrated-hydrated liposomes composed of egg phosphatidylcholine (EPC, bilayer-forming lipid), 1,2-dioleoyl-3 trimethylammonium-propane (DOTAP, cationic lipid), 1,2-dioleoyl-*sn*-glycero-3 phosphoethanolamine (DOPE, helper lipid) in a 50:25:25 percent molar ratio. The authors demonstrated that the encapsulation process protects the DNA vaccine against incubation with sodium dodecyl sulfate (SDS) due to DNA incorporation inside the liposome lamella (Perrie & Gregoriadis, 2000), and a better immunological response was obtained with cationic liposomes compared to naked DNA.

Concerning size and polydispersity, different authors have reported that nanoparticle size is an important parameter for transfection success (Ma et al., 2007; Ogris et al., 1998; Rejman et al., 2004; Ross & Hui, 1999; Wiewrodt et al., 2002). Indeed, most of the variability in transfection procedures is a consequence of non-viral gene delivery systems with high polydispersity index values. The polydispersity index is related to the width of the particle

Technological Aspects of Scalable Processes for the

homogenation and reduction of liposome size.

aggregation, forming multilamellar liposomes (Figure 1).

Fig. 1. Unilamellar and multilamellar liposomes.

techniques, equipment, and the software used for analyzing data.

translational diffusion coefficient using the Stokes-Einstein equation:

**2. Liposomes: Structure, formation, and characterization** 

Production of Functional Liposomes for Gene Therapy 269

In this chapter, we review the top-down and bottom-up approaches for liposome production and DNA complexation aiming at gene vaccines. The technological processes are classified into strategies and the feasibility to scale transposition envisaged. The physicochemical aspects of the processes and properties of the produced liposomes are correlated, connecting product and process. In addition, we report our experience with a scalable production of previously designed cationic DNA-EPC/DOPE/DOTAP liposomes in a topdown process involving ultra-turrax and microchannel microfluidizer devices for the

In general, amphiphile molecules, when dispersed in a liquid medium, undergo internal selforganization, generating colloidal aggregates. Liposomes are formed through the aggregation of phospholipid molecules in an aqueous medium. Initially, a plain bilayer structure is formed when the relative volumes between the non-polar and polar parts of the phospholipid molecules are favorable (packing factor is close to 1) for vesiculation in closed structures. The timing of spontaneous self-aggregation is long, and in technological processes this time is reduced by promoting vesiculation via the addition of energy to the system and remotion of the organic solvent or detergent in which the lipids were initially dispersed (Gregoriadis, 1990; Lasic, 1993). Primary aggregation generates unilamellar liposomes, which undergo secondary

The main physico-chemical characterization of cationic liposomes for gene vaccines includes average hydrodynamic diameter and size distribution, zeta potential, and morphology. These characterizations have been well described in the modern literature, including

Regarding the average hydrodynamic diameter and size distribution, photon correlation spectroscopy (PCS) and dynamic laser light scattering using a Ne-He laser are generally used for measurements at various incidence angles. Particle diameter is calculated from the

Where d(H) is the hydrodynamic diameter, D is the translational diffusion coefficient, k is Boltzmann's constant, T is the absolute temperature, and η is the viscosity. The mean diameter and size distribution are estimated by an adequate algorithm analysis. The results of the population distributions are expressed as the intensity of scattered light and automatically converted to number-weighted mean diameter and size distribution by adequate software. For more accurate size characterization, intensity and number-weighted

d(H) = (kT)/(3πηD) (1)

size distribution. High polydispersity indicates that greater population fractions are out of the optimum size range for cell transfection (Hsieh et al., 2009).

In this sense, a rational design associated with a technological process able to construct functional liposomes efficiently is needed. Otherwise, the technological processes must be scalable in order to guarantee the reproducibility of the formulation when more liposomes have to be produced for pre-clinical and clinical assays before industrial production. In addition, deep understanding of the phenomena involved, as well as the correlation among operational variables and the physico-chemical parameters, allows more precise control of the quality of the final product and a rational optimization of the process. Therefore, process and product must be strongly linked to assure the desired biological performance.

The literature reports valuable results regarding the biological performance of cationic liposomes in vaccination and gene therapy. However, to the best of our knowledge, no study has yet focused on the approach connecting design, product, and production process. We obtained promising immunological responses and a prophylactic effect against tuberculosis (TB) using cationic dehydrated-hydrated liposomes (DRV) complexed to DNA containing the hsp65 gene on the external surface (Rosada et al., 2008). Hsp65 is a heat shock protein from *M. leprae*. The hsp65 gene has been extensively studied and its biological effects evaluated for protection against and the treatment of tuberculosis (de Paula et al., 2007; Silva et al., 2005). Formulations based on the hsp65 gene have been used in different immunization strategies and the evaluation of protection against *M. tuberculosis* challenge (Souza et al., 2008).

We developed a novel and non-toxic formulation of cationic liposomes in which the hsp65 DNA vaccine was entrapped or complexed on the external surface of the cationic DRV. The formulation was used to immunize mice by intramuscular or intranasal routes. A single intranasal dose of the formulation elicited humoral and cellular immune responses that were as strong as those induced by four intramuscular doses of naked DNA in the mouse model of TB. The formulation also allowed a 16-fold reduction in the amount of DNA administered. Moreover, this formulation was demonstrated to be safe, biocompatible, stable, and easy to manufacture at low cost. We think that this strategy can be applied to human vaccination against TB in a single dose or in prime-boost protocols with a tremendous impact on controlling this neglected disease (Rosada et al., 2008).

The net positive surface charge imparted by DOTAP drives electrostatic binding to the cell membrane. The fusogenic properties of phosphatidylethanolamine promote the exchange of lipids with the endosomal membrane and delivery of DNA into the cytoplasm (Xu and Szoka, 1996). Finally, the presence of EPC provides stability to the liposomes, reduces the cytotoxicity of the cationic lipids, and delimits the diameter of liposome construction, which influences macrophage phagocytosis. For the same lipid composition, the complexation of DNA on the surface of liposomes generated more cell accessibility (non-electrostatically bound DNA) to DNA in relation to encapsulated DNA, and controlled antibody production related to DOTAP/DOPE lipoplexes (de la Torre et al., 2009). The liposomes were prepared using Bangham's method (Bangham et al., 1965) and characterized according to size, polydispersity, and zeta potential. DNA loading and availability on the external surface of liposomes were also determined (Rosada et al., 2008).

For the next pre-clinical and clinical assays, a scalable process had to be established in order to evaluate the capability of the system in scale transposition, reproducing the physico-chemical and biological properties of the previously prepared liposomes on a laboratory scale.

size distribution. High polydispersity indicates that greater population fractions are out of

In this sense, a rational design associated with a technological process able to construct functional liposomes efficiently is needed. Otherwise, the technological processes must be scalable in order to guarantee the reproducibility of the formulation when more liposomes have to be produced for pre-clinical and clinical assays before industrial production. In addition, deep understanding of the phenomena involved, as well as the correlation among operational variables and the physico-chemical parameters, allows more precise control of the quality of the final product and a rational optimization of the process. Therefore, process and product must be strongly linked to assure the desired biological

The literature reports valuable results regarding the biological performance of cationic liposomes in vaccination and gene therapy. However, to the best of our knowledge, no study has yet focused on the approach connecting design, product, and production process. We obtained promising immunological responses and a prophylactic effect against tuberculosis (TB) using cationic dehydrated-hydrated liposomes (DRV) complexed to DNA containing the hsp65 gene on the external surface (Rosada et al., 2008). Hsp65 is a heat shock protein from *M. leprae*. The hsp65 gene has been extensively studied and its biological effects evaluated for protection against and the treatment of tuberculosis (de Paula et al., 2007; Silva et al., 2005). Formulations based on the hsp65 gene have been used in different immunization strategies

and the evaluation of protection against *M. tuberculosis* challenge (Souza et al., 2008).

tremendous impact on controlling this neglected disease (Rosada et al., 2008).

liposomes were also determined (Rosada et al., 2008).

We developed a novel and non-toxic formulation of cationic liposomes in which the hsp65 DNA vaccine was entrapped or complexed on the external surface of the cationic DRV. The formulation was used to immunize mice by intramuscular or intranasal routes. A single intranasal dose of the formulation elicited humoral and cellular immune responses that were as strong as those induced by four intramuscular doses of naked DNA in the mouse model of TB. The formulation also allowed a 16-fold reduction in the amount of DNA administered. Moreover, this formulation was demonstrated to be safe, biocompatible, stable, and easy to manufacture at low cost. We think that this strategy can be applied to human vaccination against TB in a single dose or in prime-boost protocols with a

The net positive surface charge imparted by DOTAP drives electrostatic binding to the cell membrane. The fusogenic properties of phosphatidylethanolamine promote the exchange of lipids with the endosomal membrane and delivery of DNA into the cytoplasm (Xu and Szoka, 1996). Finally, the presence of EPC provides stability to the liposomes, reduces the cytotoxicity of the cationic lipids, and delimits the diameter of liposome construction, which influences macrophage phagocytosis. For the same lipid composition, the complexation of DNA on the surface of liposomes generated more cell accessibility (non-electrostatically bound DNA) to DNA in relation to encapsulated DNA, and controlled antibody production related to DOTAP/DOPE lipoplexes (de la Torre et al., 2009). The liposomes were prepared using Bangham's method (Bangham et al., 1965) and characterized according to size, polydispersity, and zeta potential. DNA loading and availability on the external surface of

For the next pre-clinical and clinical assays, a scalable process had to be established in order to evaluate the capability of the system in scale transposition, reproducing the physico-chemical

and biological properties of the previously prepared liposomes on a laboratory scale.

the optimum size range for cell transfection (Hsieh et al., 2009).

performance.

In this chapter, we review the top-down and bottom-up approaches for liposome production and DNA complexation aiming at gene vaccines. The technological processes are classified into strategies and the feasibility to scale transposition envisaged. The physicochemical aspects of the processes and properties of the produced liposomes are correlated, connecting product and process. In addition, we report our experience with a scalable production of previously designed cationic DNA-EPC/DOPE/DOTAP liposomes in a topdown process involving ultra-turrax and microchannel microfluidizer devices for the homogenation and reduction of liposome size.
