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

利用功能化胶体AFM探针，研制了原子力显微镜和共聚焦激光扫描显微镜（CLSM）集成的纳米尺度观测系统。该系统已被用于获得以下信息：(1)工程材料与活细胞之间的粘附力；(2)细胞摄取颗粒的最佳尺寸和表面特性；(3)细胞摄取和排出过程；(4)靶细胞及其邻近细胞的运动。我们的目的是利用我们开发的系统获得有效设计DOS颗粒的定量基础数据，并了解颗粒对活细胞的粘附，摄取和细胞毒性机制。首先，我们采用原子力显微镜（AFM）测量了小鼠皮肤癌细胞（B16F10）与具有不同电荷密度、亲疏水性和官能团表面特性的颗粒之间的粘附力。其次，我们对暴露于含有不同表面功能和大小的荧光纳米颗粒的培养基后的活细胞进行CLSM观察。此外，我们用台盼蓝染色法评估了荧光纳米颗粒的细胞毒性。研究发现，纳米颗粒的表面功能影响细胞的摄取区（细胞膜外表面或细胞质），从而决定细胞毒性。这些摄取区和细胞毒性受培养条件如温度和血清添加量的影响。吸收量与粘附力之间没有明确的关系，说明粘附力不能很好地解释细胞对颗粒的吸收量，因此需要考虑其他因素。在未来的研究中，我们应该对细胞膜上的受体（即膜蛋白）进行实验，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.