| Liposome has a sphere structure which is composed of phospholipid molecules. Liposome is amphiphilic because phospholipid molecule generally consists of hydrophobic tails and a hydrophilic head. To date, liposome is mainly used for drug carrier and artificial cell model. Disadvantages of conventional liposome preparation lies in non-uniform size distribution and unstability. Post processing, which will reduce the utilization rate of liposomes, is usually needed to make liposomes with uniform particle size. Electrictransfection is often used to import allogenic material to an artificial cell model to simulate real cell life activities. But conventional electricaltransfection often require high voltage, which cannot monitor dynamically the process of transfection. Therefore, invention of microfluidic chip is necessary for solving the above two problems.In this paper, a liposome preparation platform and electroporation dynamic monitoring platform based on microfluidic chip were set up which were summarized as follows:(1) The simulation and manufacture of microfluidic chip. Software such as Gambit was used to establish the internal structure model and Fluent was then used for flow field simulation. The main factors for investigation includes width of mircochannel, entrance angle and the flow rate of side channel, the central channel fluid in a mixed fluid volume ratio was set as reference indicators. Best mixing efficiency was obtained when the width of microchannel is100microns and entrance angle is90°. The critical flow rate ratio of side channel to central channel is10.(2) Prepartion of liposomes based on microfludic chip. POPC and DMPC were used for liposomes preparation. Different facts which influence the size and stability of liposome were investigated and theoretically explained. Levels such as FRR values, temperature and cholesterol which affect liposome size were investigated the It was found that Low FRR values resulted in get large particle size of the liposomes (900nm-1600nm) with a wide size distribution. In the high FRR values led to small particle size with narrow size distribution. Liposome size decreases as FRRs increase. As temperature increased within the range of20℃to50℃, liposome size decreased. However, a sudden increase of the size of the phospholipid DMPC was found at23℃, which is due to the phase transition temperature of phospholipids makes the elastic modulus of the film was increased by3to5times; The particle size of liposome was increased with the addition of cholesterol and reached the maximum value when the molar ratio cholesterol was20%. FRR values obtained by orthogonal experiments greatest impact on the liposome particle size, when FRR=4, the temperature is20℃, the addition of cholesterol is20%obtained liposome diameter of276.8±3.2nm, and uniformity better. For the liposome stability, The stability at the high concentration of liposomes higher than which produced at low concentrations; The addtion of surfactants will greatly improve the stability of liposomes without influence the particle size. DTAC complex with POPC at a molar ratio of1:10obtained the Zeta potential from13.6±0.54mV to26±1.83mV, hexadecyl phosphate potassium complex with POPC liposomes obtained the Zeta potential from-27.38±1.1mV to -32.8±0.39mV; Cholesterol added enhance the stability which at20%of the added amount of Zeta potential reaches at-44.74±0.95mV. Furthermore, use CLSM, TEM to observe the microstructure of liposomes which formation at different FRR values.(3) Real-time monitoring of electroporation experiments based on Confocal Laser Scanning Microscope. With liposomes and yeast cells as experimental subjects to achieve electroporation at low voltage. Liposome system:NBD-PE and POPC mixed at0.1%molar ratio to preparate fluorescent liposomes. At the conditions of100V, the pulse width of200ms with single pulse, at CLSM selected region of interest (ROI), observe the change in fluorescence intensity over time curve. Since the calculated theoretical transmembrane voltage is375mV, below the critical transmembrane which can breakdown the membrane, electroporation without success; Yeast cell system:a fluorescent probe labeled with membrane of yeast cells, apply the same voltage and pulse width, the fluorescence intensity did not change significantly. To explore succeed or not, with a carboxyl group of the water-soluble8μM CdSe/ZnS quantum dots of non-specific markers for yeast cells, the membrane is selected as ROI1, the pole position at the plasma membrane selected as ROI2. At the conditions of at100V, the pulse width of200ms with single pulse achieved the yesat cell electroporation. |
