(299c) The Effect of the Shear Rate On the Surface Properties of Egg-PC Liposomes

A variety of liposome preparation techniques has been developed since liposome was first prepared by the Bangham Method, in 1965. Because of the promising results obtained by the use of lipid vesicles in food and pharmaceutical industries, since the 1990's the laboratory scale production began to be insufficient to meet the demand for this type of material, being necessary the development of large scale techniques (GREGORIADIS, 1993; LASIC, 1993). High-pressure treatments as homogenizers or multichannel microfluidization have been taken place since the mid 80's as an easy, clean and scalable processing leading to the homogeneous and nanometric sizes of liposomes required for specific applications (MAYHEW, et al., 1987; TALSMA, ÖZER, VAN BLOOIS, & CROMMELIN, 1989). In addition, these techniques allow to processing a large volume of liposomes without the use of sonication, detergents or organic solvents (THOMPSON & SINGH, 2006). However, in order to reduce the time, number of cycles and energy consumption, a prior step of homogenization is required. Furthermore, the previous mixture step is critical to reproducing the physical-chemical properties of particles, when multicomponent systems such as mixture of functional lipids or polymer additives are used. The objective of this work is to systematically investigate the effects of the shear rate and flow rate on the size, polidispersity and zeta potencial of liposomes composed of Egg-phosphatidylcholine. The effects were produced from two levels of shear, using a Turrax equipment (2,560 to 21,430 1/s), and a mechanical disperser with cowles impeller (1,000 to 2,530 1/s). In order to reduce the total number of experiments and statistically optimize the processes variables, we have used factorial design. This tool has proven to be a useful instrument of analysis in the study of the response surface (NETO, SCARMINIO, & BRUNS, 2003). Experimental

The liposome dispersion was obtained by pouring a solution of EPC Degussa with 60% purity diluted in ethanol (400 mM) into aqueous medium at room temperature. The lipid solution was added into the aqueous phase through a peristaltic pump with pre-determined flow rate. The shear rate was adjusted in rpm, using an Ultra-Turrax IKA T25 (Ika Works) and mechanical disperser RW20 (Ika Labortchnik) with cowles impeller. The Figure 1 shows a scheme of the experimental set up.

Figure  SEQ Figure \* ARABIC 1 Schematic diagram of experimental set up (1) High shear blender, (2) aqueous solution, (3) lipid solution and (4) peristaltic pump.

The liposomes were characterized by hydrodynamic diameter, distribution and zeta potential using a Zetasizer Nano ZS (Malvern Instruments Ltd.). The mean diameter and polidispersity were determined by dynamic light scattering.          The experiments were planned according to the levels of the Table 1.

Table  SEQ Table \* ARABIC 1 Experimental planning for each type of dispersor

Results

Figure 2 shows the effects of the shear rate and flow rate on the mean diameter, polididispersity and zeta potential of the liposomes prepared using the Turrax disperser. The Figure 3 presents the behavior of the samples when the lipid dispersion was processed through the mechanical impeller.

Figure  SEQ Figure \* ARABIC 2 Response surfaces for dispersion in Turrax. (A) Mean hydrodynamic diameter, (B) polidispersity and (C) zeta potential

Figure  SEQ Figure \* ARABIC 3 Response surfaces for dispersion mechanical disperser with cowls impeller. (A) Mean hydrodynamic diameter, (B) polidispersity and (C) zeta potential

As can be seen in Figure 2, the liposomes obtained with dispersion using Turrax had mean hydrodynamic diameters varying from 250 nm to 550 nm, polydispersity from 0.30 to 0.58 and zeta potential from -57 mV to -49 mV. Moreover, in lower shear rates it is statistically stated that the influence of lipid phase flow rate in the mean diameter and polydispersity is more emphasized. Considering that the best particle conditions are with lower diameter and polydispersity, the best operational situation for the dispersion with Turrax is with low inlet lipid solution flow rate and high shear rate.

The parameters were analyzed after one day and again after three days of the preparation, showing no statistical difference between the two measurements. This is an indicative that the process is able to form liposomes with certain stability. However, in order to obtain more information, it is necessary to valuate these data after a longer period of time.

For the same levels of shear and flow rate, using the mechanical disperser with cowles impeller (Figure 3), the liposomes were formed with a larger mean hydrodynamic diameter (430 nm to 700 nm), polydispersity (0.50 to 0.68) and absolute zeta potential (-70 mV to -53 mV). Although the tendencies of each parameter's effect over the responses were similar for both dispersion systems, the Turrax disperser resulted in liposomes with more interesting characteristics for a solution to be latter processed in a microfluidizer.

In both cases, the effects of thermal dissipation during dispersion may alter the liposome packing. This result has direct influence in the particle's zeta potential. Additional research will be taken place with the purpose of studying this effect.

Conclusions

It is possible to obtain Egg-PC liposomes in nanometric scale (250 nm) with relative narrow polydispersity (0.30) from a simple process and using conventional equipments, only by controlling the shear rate of the disperser and the lipid phase inlet flow rate.

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