Fei tecnai g2 f20 twin tem

The liposome structures were examined by the FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). A 200-mesh copper grid-supported holey carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA) was glow-discharged in an argon/oxygen atmosphere for 15 s. Four microliters of the liposome sample containing ~0.5 mM lipids were pipetted onto the copper grid, paper-blotted for 3 s in a 100% humidified chamber at 4 °C and plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot system (FEI, Hillsboro, OR, USA). The grids were stored in liquid nitrogen until mounted for imaging. EM imaging was conducted in bright-field mode at an operating voltage of 200 kV. Images were recorded at a defocus value of ~1.8 μm under low-dose exposures (25–30 e/Ų) with a 4k $\times$ 4k charge-coupled device camera (Glatan, Pleasanton, CA, USA) at a magnification of 50,000×. All experiments were carried out at the Academia Sinica Cryo-EM Facility (Taipei, Taiwan).

The structures of LNC-Dox were examined using an FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). Samples were prepared for cryo-EM on an FEI Vitrobot by pipetting 4 μL of the reaction mixture containing ~0.5 mM lipids onto a 200-mesh perforated carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA), blotted for 3 s, and stored in liquid nitrogen until imaging. Images were taken at a defocus value of ~1.8 μm under low-dose exposures (25–30 e/Å2) at 50,000-fold magnification on a FEI Tecnai F20, operating at 200 kV. All experiments were carried out at the Academia Sinica Cryo-EM Facility (Taipei, Taiwan).

The FT-IR spectra of the as-fabricated biochar were collected on a Spectrum Two L1600300 spectrometer (PerkinElmer Inc., Waltham, MA, USA). The Raman spectra of the biochar were recorded on a DXR Raman Microscope Laser Raman spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The XRD patterns of all samples were performed on a MiniFlex600 diffractometer (Rigaku Corporation, Tokyo, Japan) in a range of 10° to 80°. The surface chemical compositions of the as-fabricated biochar were determined by an Escalab 250Xi XPS (Thermo Fisher Scientific Inc., Waltham, MA, USA) characterization. The STA-449F3 thermal balance analyzer (NETZSCH Inc., Selb, Bavaria, Germany) was used to obtain the TG curves of the as-fabricated biochar. The surface morphology of biochars were recorded by SEM images on a REGULUS 8230 SEM (Hitachi Co., Tokyo, Japan) and TEM images on a FEI Tecnai G2 F20-TWIN TEM (FEI Co., Hillsboro, OR, USA). The micromeritics TRISTAR II3020 surface area analyzer (Micromeritics Instrument, Norcross, GA, USA) was used to determine the BET surface area of biochars. Zeta potentials of different pH solutions were recorded on a Zetasizer Nano ZS90 zeta potential analyzer (Malvern Panalytical, Malvern, UK). The total amount of heavy metal ions was determined by an ICP-OES (ICAP7400, BRE0002948, Thermo-Fisher Scientific Inc., Waltham, MA, USA).