Em 002b

The X-ray diffraction
(XRD) patterns were acquired with a PANalytical X’Pert PRO
diffractometer with Cu Kα radiation. The microstructures of
the samples were examined by scanning electron microscopy (SEM; JSM-7600F,
JEOL). The bright-field and lattice images and selected-area electron
diffraction (SAED) patterns were observed by transmission electron
microscopy (TEM; EM-002B, TOPCON). The ultraviolet–visible
(UV–Vis) diffuse reflectance spectra were recorded on an Evolution
220 UV–vis spectrometer (Thermo Fisher Scientific).

Cerebellum tissue specimens obtained from 27 mice (3 per stage of WT, AMS and Nna1 KO mice) were fixed in 2% glutaraldehyde overnight at 4 °C and rinsed with 0.2 M PB buffer for 20 min. Samples were dehydrated in a graded series of acetone (25%, 50%, 75%, 100%) before being embedded in epoxy resin (TAAB medium grade) and polymerized overnight at 60 °C. Sections were cut on a Leica EM UC7 ultramicrotome. First, semithin sections (0.5 μm) were stained with toluidine blue for light microscopy to identify the area of interest and confirm the orientation of the tissue. Ultrathin sections (70 nm) were then transferred to copper grids, stained with uranyl acetate and lead citrate and examined on a Topcon EM-002B (Tokyo, Japan) transmission electron microscope. Images were captured at ×10,000 magnification.

The structure and morphology of the prepared LM nanocapsules were observed using
high-resolution TEM (EM-002B; Topcon, Tokyo, Japan) with an accelerating voltage
of 120 kV. A small droplet of sample placed on a grid or
10 μl of sample solution (LM concentration:
100 μg ml⁻¹) were
irradiated using a fibre-coupled CW laser at 785 nm for
3 min (spot diameter, ∼4 mm; maximum power:
1 W,
∼80 mW mm⁻²;
BRM-785-1.0-100-0.22-SMA; B&W Tek, Newark, DE, USA). The polymer shell
structure of nanocapsules and STEM/EDS mapping were performed by Nanoscience Co.
in Evans Analytical Group Company, Inc. (Tokyo, Japan). The samples were imaged
with a FEI Tecnai TF-20 FEG/TEM operated at 200 kV in bright-field
TEM mode, high-resolution (HR) TEM mode, and high-angle annular dark-field
(HAADF) STEM mode. The STEM probe size was 1–2 nm nominal
diameter. EDS mapping were acquired on Oxford INCA, Bruker Quantax EDS
system.
The hydrodynamic diameter of LM nanocapsules was examined by DLS (Photal
FPAR-1,000; Otsuka Electronics, Osaka, Japan). DLS diagram of laser-induced LM
nanocapsules was also measured. A 100 μl of sample solution
(LM concentration:
100 μg ml⁻¹) was
irradiated using a fibre-coupled CW laser (maximum power: 1 W,
∼80 mW mm⁻²) at
785 nm for 1 h before DLS measurements.
The concentration of LM and carmofur in nanocapsules was estimated with a
ultraviolet–visible–NIR spectrophotometer (V-730 BIO; Jasco,
Tokyo, Japan).

The structure and morphology of the prepared ND-SPs were visualized using a high-resolution transmission electron microscope (TEM) (EM-002B, Topcon, Tokyo, Japan) at an accelerating voltage of 120 kV.
The hydrodynamic diameter of the ND-SPs was determined via dynamic light scattering (DLS) (Photal FPAR-1000, Otsuka Electronics, Osaka, Japan).
A UV-Vis-NIR spectrophotometer (V-730 BIO, Jasco, Tokyo, Japan) was used to measure the spectral profiles and concentrations of the ND-SPs and CPT complexes.
Fourier transform infrared (FTIR) spectroscopy (Spectrum One, PerkinElmer, Yokohama, Japan) analysis was carried out to identify the presence of alkyl chains on the ND surface.