Optimization Of The Preparation Methods Of Giant Vesicles On ITO Glass Device

Liposomes are enclosed vesicles formed by the spontaneous arrangement and encapsulation of the phospholipid molecules. Among them, the liposomes with diameters of 1 to 200 mm are called as giant vesicles. Giant vesicles have been widely used because of its special characteristics. They have huge sizes and are non-toxic, so they can be used as good drug carriers. Furthermore, their str uctures and membranes are similar to the cell and cell membrane, so they can also be utilized as models to study their properties. In the studies of giant vesicles, the preparation has always been a topic of concern, but on a whole, the exploration of this aspect is still at its preliminary stages, the preparation efficiency is still not good enough. Therefore, this paper will focus on the optimization of the existing preparation methods and try to improve the preparation efficiency by exploring the effects of various factors on the formation process of giant vesicles. The aim is to obtain a large number of well-distributed giant vesicles on a simple “sandwich” device and explore to the preparation of giant vesicles under physiological condition.Firstly, giant vesicles were prepared on the original chip made in our lab. According to the experimental results of bubble formation and the impact of water flow on the lipid film, another “sandwich” device based on ITO glass was designed and manufactured. The experimental results in this new chip showed that some existing problems in the original chip can be avoided effectively.Electroformation of giant vesicles was carried out on the newly developed chip, and the impact of different electrical parameters(intensit y and frequency) on the electroformation process was explored. The experimental results showed that the electric field intensity and the electric field frequency acted in conjunction to influence the electroformation of giant vesicles. When the electric field intensity was low, the effect of the frequency on the formation of giant vesicles was unapparent. When the electric field intensity was high, the effect of the frequency was much obvious, but no matter what the frequency was there were always some giant vesicles deformed and broken out because of the high intensity. Only when the intensity was appropriate, the effect of frequency can be observed evidently, and formed giant vesicles were relatively spherical. Under an appropriate electric field intensity, when increasing the frequency, the amount of giant vesicles formed in the chamber increased and the size of the vesicles gradually decreased and tended to be more and more uniform. This trend would be maintained until a specific frequency, and a largest number of giant vesicles with narrow size distribution were obtained at this frequency. When the frequency continuously increased over this specific value, the amount of giant vesicles gradually reduced to the stability while their sizes and distributions were not significantly changed. Thus, the preparation effect of the giant vesicles reached best at this specific frequency, and in this paper the value of the specific frequency is 11 k Hz. Theoretical analysis and model simulation indicated that the effect of electric field on the lipid film was realized by the direct effect of the dielectrophoretic effect(DEPE) and the indirect effect of the electrohydrodynamic effect(EHE). And the mutual effect of both can be maximized by the characteristic frequency of the system. The value of the characteristic frequency was theoretically calculated(13.3 k Hz) and found to be very close to the optimal frequency(11 k Hz) obtained by the experiments. This result demonstrated that it might be the maximization of the electric field lead to the optimization of the giant vesicles preparation. Therefore, for a given electroformation system, even a simple device such as the “sandwich” chip used here, large amount of well-distributed giant vesicles can be obtained by optimizing the electrical parameters. In this paper, when choosing the optimal electrical parameters(11 k Hz frequency and 5 V/mm intensity), the yield of giant vesicles could reach 800/chamber, and the diameters of most vesicles(> 70%) were 40-60 mm.In order to further improve the yield of giant vesicles, a new lipid film formation method was explored. Different from the previous bottom- forming method, this new method formed lipid film on the top glass slide of the “sandwich” device. Experimental results demonstrated that the top-forming method was indeed an effective one by changing the external force effect on the lipid film, a higher preparation efficiency was obtained(more than 1000/chamber). Through forming lipid films both on the bottom and top glass slide of the chip, plate electrodes of the “sandwich” device were fully utilized and the yield of the giant vesicles could reach 2000/chamber. In this way, the simple device showed its unique potential in giant vesicles preparation that other structures do not have.A series of experiments were carried out to study the effect of ion concentrations on the formation of giant vesicles. Experimental results and analysis indicated that ions reduced the effect of electric field and inhibited the curling and separation of the lipid film, and hence hindered the formation of giant vesicles. In order to overcome the blocking effect of ions, the external force and the lipid film properties were both improved by adopting the top- forming method and increasing the experimental temperature. A large number of giant vesicles were successfully prepared under the high ions concentration of 150 m M(more than 800/chamber), which made the foundation for the preparation of giant vesicles under the physiological condition.