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Combination analysis of stability of a nanomedicine and release of the encapsulated drug using DLS, AFM and HPLC.AmBisome aggregation or collapse and release of amphotericin B occurred upon addition of more than 75% methanol or dilution with buffer solution around pH 4.Physico-chemical properties analyses by different.Methods and the integration of these data are required for understanding the behavior of nanoparticles.Much attention has been paid to nanoparticle-based drug delivery systems to selectively deliver drugs to target organs and tissues. In this study, AmBisome, an antimycotic nanomedicine containing amphotericin B, was chosen as a typical nanomedicine, and analyzed using dynamic light scattering (DLS), an easy method to measure size, and size distribution, atomic force microscopy (AFM) and high-performance liquid chromatography (HPLC). AFM allowed the analysis of shape, besides a detailed size and size distribution. HPLC is useful to quantify the released amount of amphotericin B as it succeeded in a rapid separation of AmBisome and free amphotericin B. The aggregation or collapse of AmBisome and release of amphotericin B occurred upon 75% methanol addition or dilution with buffer around pH 4. Therefore, it is important to analyze nanomedicines using multiple analytical methods, and to integrate them for the quality and quantity evaluation of nanomedicines.