Development of nano-scale observation system for adhesion and uptake of potholes on/into living cells

我们开发了一个集成的原子力显微镜和共聚焦激光扫描显微镜（CLSM）与功能化胶体AFM探针的纳米尺度观察系统。该系统已被用于获得以下信息：（1）工程材料和活细胞之间的粘附力;（2）细胞摄取的颗粒的最佳尺寸和表面性质;（3）细胞摄取和放电的过程;（4）靶细胞及其邻近细胞的运动。首先，利用原子力显微镜（AFM）测量了小鼠皮肤癌细胞（B16 F10）与具有不同表面性质（电荷密度、亲水/疏水性和官能团）的DOS颗粒之间的粘附力。其次，我们进行CLSM观察暴露于包括具有各种表面功能和尺寸的荧光纳米颗粒的培养基后的活细胞。同时，我们还利用台盼蓝染色法对纳米颗粒的细胞毒性进行了评价，发现纳米颗粒的表面功能性影响了细胞的摄取区域（细胞膜外表面或细胞质），从而决定了纳米颗粒的细胞毒性。这些摄取区域和细胞毒性受培养条件如温度和血清添加的影响。细胞摄取量与粘附力之间没有明确的关系，表明粘附力不能很好地解释细胞对颗粒的摄取，因此应考虑其他因素。膜蛋白）▲以了解细胞摄取颗粒的详细机制。

We developed an integrated nano-scale observation system of an Atomic Force Microscope and a Confocal Laser Scanning Microscope (CLSM) with the functionalized colloid AFM probes. This system has been used to obtain the following information: (1) adhesion forces between engineered materials and living cells; (2) optimal size and surface property of particles for cellular uptake; (3) processes of cellular uptake and discharge; (4) movement of the target cells and their neighboring cells. Our purpose is to obtain the quantitative fundamental data for efficient design for DOS particles using our developed system and to understand the mechanisms of adhesion, uptake, and cytotoxicity of particles for living cells.First, we carried out AFM measurement of the adhesion forces between mouse skin cancer cells (B16F10) and particles with different surface properties of charge density, hydorophilicity/hydrophobicity, and functional group. Second, we performed CLSM observation of the living cells after exposure to culture media including fluorescent nanoparticles with various surface functionalities and sizes. Also, we assessed the cytotoxicity of the fluorescent nanoparticles using the trypan blue dye method.It was found that the surface functionality of nanoparticles affects the uptake region of cells (the outer surface of cell membrane or the cytoplasm) to determine the cytotoxicity. These uptake region and cytotoxicity were affected by the culture conditions such as temperature and serum addition. There were no clear relationship between the uptake amount and the adhesion forces, indicating that the cellular uptake of particles is not satisfactorily explained by the adhesion forces and additional factor(s) should be therefore took into account.In the future study, we should carry out the experiments focusing on the receptors residing in/on cell membrane (i.e., the membrane proteins)▲to understand the detailed mechanisms of cellular uptake of particles.