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

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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.