4k charge coupled device camera

The structure of fragment species of Dox-MBs was observed using the cryo-EM imaging performed on the FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). Initially, a 200-mesh copper grid-supported holey carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA) was placed in an argon/oxygen atmosphere and was surface-modified by glow-discharge for 15 s. A 4 μl sample was added onto the copper grid blotted for 3 s in a 100 % humidified chamber at 4 °C. Subsequently, it was flash-frozen in liquid ethane that had been cooled by liquid nitrogen using the Vitrobot sample plunger system (FEI, Hillsboro, OR, USA). The prepared copper grid was stored in liquid nitrogen until imaging. Imaging was conducted in the bright-field mode for cryo-EM with an operating voltage of 200 kV. The images were captured at a magnification of 50,000 × with a 4 k × 4 k charge-coupled device camera (Gatan, Pleasanton, CA, USA).
To investigate the size distribution of fragment species in LDox-MBs and HDox-MBs, the samples with and without US were centrifuged, and the bottom layer solution was analyzed. Particle size and concentration were measured using dynamic light scattering (DLS, model ZetaSizer 3000, Malvern, Worcs., UK). Before analysis, the bottom layer solution was diluted 1000-fold with PBS to achieve concentrations between 10⁶ and 10⁹ particles/ml.