**6.1.1 Statistical analysis**

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, respectively.

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 before characterization.

Technological Aspects of Scalable Processes for the

rate were significant for polydispersity.

(A3,B3) zeta potential.

200 nm.

Production of Functional Liposomes for Gene Therapy 283

Data obtained using ultra-turrax showed a significant influence of the shear and feed flow rates, as well as their interactions, on the mean diameter and polydispersity (Figure 7B1,B2). However, these variables had no significant effect on the zeta potential (Figure 7B3). The mean diameter was influenced more by shear rate, whereas both shear rate and feed flow

Fig. 7. Pareto´s graphic for liposome production using (A) Caules and (B) Ultra-Turrax*®*  stirrers. (A1,B1) mean diameter hidrodinâmico médio cumulativo, (A2,B2) polydispersity,

The results show that the shear rate range of the Caules stirrer did not comminute the aggregates less than 600 nm in size, but it destroyed the progressive aggregations, influencing the polydispersity and zeta potential. However, the higher shear rates provided by the ultra-turrax better control phospholipid aggregation, generating smaller liposomes. Control is strongly dependent on the intensity of shear, and sizes in the range of 200 nm were obtained at the superior shear rate limit (21530 s-1) provided by the equipment. Feed flow rate influence mainly occurs at the inferior shear rate limit (2860 s-1). Though the shear rate range for ultra-turrax interferes with primary liposome aggregation, the zeta potential is not influenced. Therefore, for egg lecithin (300 mM concentration) liposomes, the shear rate range between 1000 and 21430 s-1 delimits the liposome size from approximately 600 to

*Mean diameter -* Additional data allowed the construction of the curve presented in Figure 8A. The curve shows a clear relationship between liposome comminution and shear rate, with the mean diameter exponentially decaying with applied shear rate. The error bars are higher at lower shear rates due to the poor homogenization of liposomes provided by the

The microchannel microfluidizer, working in the pressure range of 200 to 1500 bars, provided shear rates in the range of 2×105 to 6×105 s-1 .The data from the microchannel microfluidizer were obtained for pre-treated liposomes using Ultra-Turrax at shear and feed flow rates of 5600 to 24000 s-1 and 0.09 to 0.96 mL.s-1, respectively. The results show that the pre-formed liposomes reached the nanometric range (100 nm) in only one passage using

This comminution behavior agrees with the results reported by Diat et al. (1993a). Through a balance between elastic and viscous forces in the liposomes, the mean diameter is reduced

Ultra-Turrax and the high shear rate range of the microchannel microfluidizer.

according to the square root of the applied shear rate (Equation 21).

**6.1.3 Effects of the shear rate on the liposome properties** 

lower pumping capabilities of the mechanical systems.

Fig. 6. Scheme of the experimental set-up used for liposome preparation using (A) Ultra-Turrax® or Caules mechanical stirrer. 1) Tank containing the lipid dispersion in ethanol; 2) peristaltic pump; 3) tank with four fins containing (150 mL) water; 4) Ultra-Turrax® or Caules mechanical stirrer. (B) Microchannel microfluidizer system processed the samples from the Ultra-Turrax®.

#### **6.1.2 Significance of shear and feed flow rates**

The effects of the shear rate and feed flow rate on the mean diameter, polydispersity, and zeta potential are presented in Table 2 and in terms of the significance of the independent variables and their interaction in the Pareto graphics in Figure 8. For the Caules type stirrer, the effects of shear rate and feed flow rate on the mean diameter of liposomes were not significant (Figure 7A1). The shear rate was significant for polydispersity (Figure 7A2), and both variables were significant for zeta potential (Figure 7A3). The interactions between the variables were not significant for the three response variables.


Table 2. (A,B) Physico-chemical properties of liposomes as a function of the operational variables.

Fig. 6. Scheme of the experimental set-up used for liposome preparation using (A) Ultra-Turrax® or Caules mechanical stirrer. 1) Tank containing the lipid dispersion in ethanol; 2) peristaltic pump; 3) tank with four fins containing (150 mL) water; 4) Ultra-Turrax® or Caules mechanical stirrer. (B) Microchannel microfluidizer system processed the samples

The effects of the shear rate and feed flow rate on the mean diameter, polydispersity, and zeta potential are presented in Table 2 and in terms of the significance of the independent variables and their interaction in the Pareto graphics in Figure 8. For the Caules type stirrer, the effects of shear rate and feed flow rate on the mean diameter of liposomes were not significant (Figure 7A1). The shear rate was significant for polydispersity (Figure 7A2), and both variables were significant for zeta potential (Figure 7A3). The interactions between the

> -0 + 2,860 12,140 21,430 0.09 0.54 0.96


1 2 Diameter (nm) Polydispersity Zeta Potential (mV) -1 -1 626.17 0.65 -55.00 1 -1 414.87 0.51 -71.27 -1 1 616.47 0.68 -57.50 1 1 446.77 0.51 -52.60 0 0 582.77 0.65 -57.27 0 0 642.67 0.58 -55.80 0 0 567.40 0.62 -56.00

1 2 Diameter (nm) Polydispersity Zeta Potential (mV) -1 -1 386.10 0.37 -53.73 1 -1 254.73 0.31 -56.33 -1 1 550.20 0.58 -53.73 1 1 246.50 0.35 -48.57 0 0 374.83 0.47 -55.90 0 0 359.00 0.36 -54.60 0 0 358.90 0.45 -54.40

Table 2. (A,B) Physico-chemical properties of liposomes as a function of the operational

from the Ultra-Turrax®.

Table A

Factors

Table B

Factors

variables.

**6.1.2 Significance of shear and feed flow rates** 

**1**: Shear rate (s-1

Experiment 

1

**1**: Shear rate (s-1)

7

5

3

2

7

6 4 5

Experiment F t <sup>1</sup>

**2**: Inlet lipid solution flow rate (mL/s)

variables were not significant for the three response variables.

) **2**: Inlet lipid solution flow rate (mL/s) Data obtained using ultra-turrax showed a significant influence of the shear and feed flow rates, as well as their interactions, on the mean diameter and polydispersity (Figure 7B1,B2). However, these variables had no significant effect on the zeta potential (Figure 7B3). The mean diameter was influenced more by shear rate, whereas both shear rate and feed flow rate were significant for polydispersity.

Fig. 7. Pareto´s graphic for liposome production using (A) Caules and (B) Ultra-Turrax*®*  stirrers. (A1,B1) mean diameter hidrodinâmico médio cumulativo, (A2,B2) polydispersity, (A3,B3) zeta potential.

The results show that the shear rate range of the Caules stirrer did not comminute the aggregates less than 600 nm in size, but it destroyed the progressive aggregations, influencing the polydispersity and zeta potential. However, the higher shear rates provided by the ultra-turrax better control phospholipid aggregation, generating smaller liposomes. Control is strongly dependent on the intensity of shear, and sizes in the range of 200 nm were obtained at the superior shear rate limit (21530 s-1) provided by the equipment. Feed flow rate influence mainly occurs at the inferior shear rate limit (2860 s-1). Though the shear rate range for ultra-turrax interferes with primary liposome aggregation, the zeta potential is not influenced. Therefore, for egg lecithin (300 mM concentration) liposomes, the shear rate range between 1000 and 21430 s-1 delimits the liposome size from approximately 600 to 200 nm.
