(98ad) Intermittent Gas/Liquid Interfaces in a Micro-Channel for Liposome Production

Liposomes produced from lipids have been studied as promising materials for drug delivery systems, since the liposomes are relatively non-toxic and biodegradable. Liposomes can keep drug insides and deliver the drug to tumor cells [1]. For using liposomes as a drug carrier, the size of liposomes and the homogeneity in their structure are the most important characteristics because they govern the stability and drug-release properties in the human body [2]. For example, liposomes whose diameter is less than 50 nm can be egested by kidney or caught by healthy cells, and liposomes whose diameter is larger than 200 nm can be eliminated by a liver. Thus, the sizes of 50 to 200 nm of liposome are suitable as a drug carrier to tumor cells. Therefore, in the production of liposomal formulations, the control of these characteristics is the main issue, and the reliable and cost-effective production method is common concern.

Bangham method is the most common technique to form liposomes [3]. Firstly, a liposomal lipid such as egg yolk lecithin is spread on the inner surface of a flask, and then an aqueous solution of a drug is added. This flask is shaken or churned to cause shear stresses, which peel off the thin layer of lipid and help to encapsulate the small droplet of aqueous solution with the lipid bilayer. Here, liposomes can be obtained. In this method, however, it is difficult to achieve a homogeneous and constant field of fluid stress, and therefore the resulting liposomes often have a variety of sizes and structures. This is disadvantageous because a screening process is necessary to obtain the liposomes of a desired size and structure, and consequently the yield becomes low. There are other some methods proposed to produce liposomes, but all of them are still required to be improved. For example, many methods use organic solvent that is harmful for human body, and many methods cannot control the size of the liposomes [4, 5].

In our previous studies [6], we proposed a micro-tube production system, which is a kind of micro-reactor [7]. In this method, a thin lipid film attached to the inner wall of a micro-tube as the first step. As the second step, the aqueous solution to be encapsulated in liposomes flows in the micro-tube. Indeed, this system achieved to obtain homogeneous liposomes compared with Bangham’s method. The peak size of liposomes was determined by Reynolds number. The mechanism of liposome formation of this method is as follows. First, lipids on the wall of the micro-tube adsorb to the head of the flow, that is, the liquid/gas interface. Second, rod-like micelles were generated at the interface due to shear stresses caused by flow circulation in the flow close to the surface. After that, the rod-like micelles were separated to spherical liposomes. Thus, not the film of lipids but the gas/liquid interface catching lipids was confirmed only to be necessary for liposomes production [7].

In this study, a novel technique of liposome production using a gas/liquid interface in a micro-channel has been suggested. By this method, the liposomes can be obtained without harmful organic solvents and the miniaturization of liposome production can be realized.

 As sample materials, egg yolk lecithin of 0.1 g as phospholipids is dispersed in 50 ml of deionized water by using ultrasonic. T-shape micro channel was used for experiments. A main channel has 400 mm width and 250 mm depth. A branch channel has 150 mm width and 250 mm depth. The main channel was used to flow dispersed phospholipids solution, and the branched channel was used to flow air. A syringe driver controlled the flow rate. The solution rate was changed from 0.0125 to 0.100 ml/min, and the air rate was changed from 0.025 to 0.200 ml/min. The flow condition of gas/liquid flow under each flow rate was observed. The effluent including liposomes has been recovered and analyzed. The effects of the flow velocity on the liposome size and on the yield of liposomes were investigated. The diameter of liposomes was analyzed with a dynamic light scattering system of Zetasizer NANO (Malvern Instruments Ltd.). The velocity of the gas/liquid interface was measured from recorded movies.

The apparent velocity determined from liquid and gas flow was the same as the real interface velocity measured by the movies, when the flow rate was low. However, the real flow rate calculated by the velocity of the gas/liquid interface in the recording movies became smaller than the apparent velocity. This implies the liquid slip through between the wall of the tube and gas column, or the gas was compressed. This mechanism was tried to be confirmed by observing the length of liquid column and the gas column under the real velocities.

The diameter of the liposomes determined as a peak diameter decreased with increase of the real flow velocities.  The peak diameter divided by hydraulic diameter was plotted to Reynold’s numbers defined by the hydraulic diameter of the micro-channel and water viscosity. The value of the peak diameter divided by the hydraulic diameter in the present study was compared to the previous data [8]. When the liquid flow rate was fixed at 0.0125 ml/min and when the gas flow rates were 0.025 and 0.5 ml/min, the value was fit to the previous data. On the other hand, when the gas flow rates were 0.0125 and 0.1 ml/min, the value did not agree with the previous data. This result indicates that the suitable length of the liquid column to form liposome exists.  Lower gas flow rate leads to the formation of too short liquid column and higher gas flow rate makes too long liquid column for liposome production. The reasonable explanation we propose here is that too short liquid column cannot generate enough shear to make rod-like micelles and too long liquid column prohibit adsorption of lipids. It is concluded that there exists the optimum condition on the gas/liquid flow rate for producing suitable liposome in the present method.

[1] R. Schwendener, Bio-Applications of Nanoparticles Advances in Experimental Medicine and Biology, 620, 117 (2007)

[2] J. Zhu, F. Yan, Z. Guo, R. E. Marchant, Journal of Colloid and Interface Science, 289, 542 (2005)

[3] A. D. Bangham, Journal of Molecular Biology, 13, 238 (1965)

[4] A. Jahn, W. N. Vreeland, D. L. DeVoe, L. E. Locascio, M. Gaitan, Langmuir, 23, 6289 (2007)

[5] L. Lesoin, C. Crampon, O. Boutin, E. Badens, The Journal of Supercritical Fluids, 57, 162 (2011)

[6] H. Suzuki, J. Hamamura, T. Katsuda, Y. Komoda, S. Katoh, H. Usui, Journal of Chemical Engineering Japan, 41, 739 (2008)

[7] K. Mae, Chemical Engineering Science, 62, 4842 (2005)

[8] A. Fujiwara, H. Suzuki, T. Katsuda, Y. Komoda, The 13^th Asia Pacific Confederation of Chemical Engineering Congress, 10388 (2010)