基于微流控芯片的脂质体制备及电穿孔实验

发布时间：2018-05-05 18:21

本文选题：微流控芯片 + 脂质体　；参考：《浙江工商大学》2015年硕士论文

【摘要】：脂质体是磷脂分子有序排列闭合形成内腔为空心的圆球型聚集体。由于磷脂的分子结构包括亲水的头基和疏水的长链,故形成的脂质体同样具有两亲性。其内部空腔可加载亲水性的物质,疏水链之间加载亲油性的物质。目前脂质体的应用主要有以下两个方面：药物载体和人工细胞模型。传统制备方法得到的脂质体粒径不均匀,稳定性较差,需要通过二次处理才能得到粒径均一的脂质体,但这会降低脂质体的利用率；作为人工细胞模型,常用电转染的方法导入外源物质,用以模拟真实细胞的生命活动。但是常规的电转染往往需要较高的电压,且不能动态监测转染过程。微流控芯片的产生为上述两个问题的解决提供了途径。本课题以微流控芯片为主体,搭建了脂质体制备平台和电穿孔动态监测平台。得到的研究结果如下： (1)微流控芯片的仿真模拟与制作。应用Gambit建立芯片内部的结构模型,再用Flunet进行流场模拟。主要考察的因素有微通道的宽度,入口角度以及侧通道的流速。以中央通道流体在混合流体所占的体积比例为参考指标,得出在微通道的宽度为100μm,入口角度为90°时流体的混合效率最好,侧通道与中央通道的临界流速比为10。 (2)基于微流控聚焦力学的方法制备脂质体。以1-棕榈酰基-2-油酰-SN-甘油-3-磷酰胆碱(POPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)为原料,分别考察了不同因素对形成的脂质体粒径及稳定性的影响,并用理论公式予以解释说明。在脂质体粒径方面,考察了侧通道与中央通道体积流速比FRR值、温度及胆固醇含量的影响：低FRR值时,得到的脂质体粒径较大(900nm-1600nm)但其粒径分布较宽。而在高FRR值时,得到的粒径较小,且多分散性较好。脂质体粒径随着FRR的增加而减小；在20℃～50℃区间内,随着温度的上升脂质体粒径有下降的趋势。但DMPC磷脂在230C时粒径突然增大,这是由于达到了磷脂相变温度使得膜的弹性模量增大了3-5倍；胆固醇的添加量使得粒径增大,且在摩尔比为20%的粒径最大。通过正交实验得到FRR值对脂质体粒径的影响最大,当FRR=4、温度为20℃、胆固醇添加量为20%时得到的脂质体粒径为276.8±3.2nm,且均一性较好。在脂质体稳定性方面：高浓度磷脂形成的脂质体稳定性高于低浓度的；表面活性剂的添加在不影响粒径的前提下大大提高了脂质体的稳定性,其中阳离子表面活性剂十二烷基三甲基氯化铵与POPC磷脂按摩尔比10%复配制得的脂质体Zeta电位在13.6±0.54mV和26±1.83mV之间,阴离子表面活性剂十六烷基磷酸钾与POPC磷脂复配得到的脂质体Zeta电位在-27.38±1.1mV和-32.8±0.39mV之间；胆固醇的添加使脂质体稳定性增强,在20%的添加量时Zeta电位达到-44.74±0.95mV。此外,还利用CLSM、TEM观察了不同FRR值下形成脂质体的微观结构。 (3)基于激光共聚焦实时监测的电穿孔实验。以脂质体和酵母细胞为实验对象实现低电压下的电穿孔。脂质体体系：NBD-PE和POPC以0.1%的摩尔比制备形成荧光脂质体,在100V、脉冲宽度为200ms、脉冲形式为单脉冲的条件下,在CLSM下选定ROI,观察荧光强度随时间变化曲线。由计算得出的理论跨膜电压是375mV,低于击穿膜的临界跨膜电压,电穿孔没有成功；酵母细胞体系：用细胞膜红色荧光探针标记酵母细胞,施加相同的电压以及脉冲宽度,其荧光强度也没有发生明显的变化。为了探究电穿孔成功与否,用8μm的羧基水溶性CdSe/ZnS量子点非特异性标记酵母细胞,选定细胞膜为ROI1,在细胞膜极点位置为ROI2,在100V、脉冲宽度为200ms,单脉冲的条件下实现了细胞的电穿孔。
[Abstract]:Liposomes are the spherical spherical aggregates of phospholipid molecules arranged in an orderly arrangement to form a hollow inner cavity. As the molecular structure of phospholipids includes the hydrophilic head and the hydrophobic long chain, the formed liposomes also have two affinity. The internal cavity can load hydrophilic substances, and the hydrophobic chain can load the oil-based substances. There are two main aspects: the drug carrier and the artificial cell model. The traditional preparation method is not uniform in the size of the liposomes, and the stability is poor. It needs two treatments to get the homogeneous liposomes, but this will reduce the utilization rate of the liposomes. As an artificial cell model, the method of electrotransfection is commonly used to import foreign substances. Quality is used to simulate the life activities of real cells. However, conventional electrotransfection often requires high voltage and can not dynamically monitor the transfection process. The production of microfluidic chips provides a way to solve the above two problems.
The microfluidic chip is used as the main body to build a platform for preparing lipid and a dynamic monitoring platform for electroporation.
(1) simulation and fabrication of microfluidic chip. The structure model inside the chip is built with Gambit, and then the flow field is simulated with Flunet. The main factors are the width of the microchannel, the angle of the entrance and the flow velocity of the side channel. When the inlet angle is 90 degrees, the mixing efficiency is the best, and the critical velocity ratio of the side channel to the central channel is 10.. The ratio is 100 m.
(2) the liposomes were prepared based on the microfluidic focusing mechanics. The effects of different factors on the particle size and stability of the liposomes were investigated with 1- palmioyl -2- oil acyl -SN- glycerol -3- phosphachcholine (POPC) and two myrisyl phosphatidylcholine (DMPC), and the theoretical formulas were used to explain the effect of different factors on the particle size of liposomes. The effects of volume velocity ratio (FRR), temperature and cholesterol content on the side channel and central channel were investigated. At low FRR value, the size of the liposomes was larger (900nm-1600nm), but its particle size distribution was wide. At the high FRR value, the particle size was smaller and the polydispersity was better. The particle size of liposomes decreased with the increase of FRR; at 20 C to 50 C In the interval, the particle size of the liposome decreased with the increase of the temperature. But the particle size of DMPC phospholipid increased suddenly at 230C. This was due to the 3-5 times the modulus of the membrane. The addition of cholesterol made the particle size increase and the particle size was the largest in the mole ratio of 20%. The FRR value was obtained by orthogonal experiment. The effect of liposome particle size is the most. When FRR=4, temperature is 20, and the addition of cholesterol is 20%, the size of the liposome is 276.8 + 3.2NM, and the homogenization is better. In the stability of liposomes, the stability of the liposome formed by high concentration phospholipid is higher than that of low concentration; the addition of the surfactant is greatly raised on the premise of not affecting the particle size. The stability of liposomes was higher, in which the cationic surfactant twelve alkyl three methyl ammonium chloride and POPC phospholipid were massaged by 10%. The Zeta potential of the liposomes was between 13.6 + 0.54mV and 26 + 1.83mV. The Zeta potential of the anion surfactant, potassium phosphate potassium phosphate and POPC phospholipid, was -27.38 + 1.1mV and -32 .8 + 0.39mV, the addition of cholesterol enhanced the stability of liposomes, and the Zeta potential reached -44.74 + 0.95mV. at the addition of 20%. CLSM, TEM was used to observe the microstructure of liposomes under different FRR values.
(3) electroporation experiments based on laser confocal real-time monitoring. Liposomes and yeast cells are used to achieve electroporation at low voltage. Liposome system: NBD-PE and POPC are prepared to form fluorescent liposomes at a molar ratio of 0.1%. Under the condition of 100V, pulse width of 200ms and pulse form as single pulse, ROI is selected under CLSM. The calculated fluorescence intensity varies with time. The calculated theoretical cross membrane voltage is 375mV, which is lower than the critical cross membrane voltage of the breakdown membrane. The electroporation is not successful; the yeast cell system: the yeast cells are marked with the red fluorescent probe of the cell membrane, the same voltage and pulse width are applied, and the fluorescence intensity has not changed obviously. To explore the success of electroporation, the non specific yeast cells were labeled with 8 m carboxyl water-soluble CdSe/ZnS quantum dots, the cell membrane was selected as ROI1, the cell membrane pole position was ROI2, and the cell electroporation was realized under the condition of 100V, the pulse width of 200ms, and the single pulse.

【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TN492

【参考文献】