H 600 tem

A small amount of striatum tissue was cut into small pieces of 1 mm × 1 mm × 1 mm, fixed in 2.5% glutaraldehyde for 24 h, post-fixed in osmic acid for 1 h, dehydrated with graded acetone, and finally embedded with epoxy resin Epon 812. Using the ultra-microtome, the tissue was cut into 50 nm ultrathin slices, put on the copper grid and stored at 4 °C. In a clean petri dish, a drop of 3% uranyl acetate-alcohol dye liquor (pH-3.5) was added to the ultrathin slices for 30 min, rinsed in H₂O for 10 min three times, and water was drained. Using the same method, the slices were dyed by 6% lead citrate dye liquor (pH-12) for 5 min, water was drained and the sections were placed at room temperature for the ultra-structure observation under TEM (Hitachi H-600 TEM).

After the liposomes were dispersed in deionized water, their particle size, and zeta potential were analyzed using a Zetasizer Nano S (Malvern Instruments, UK). Liposome morphology was examined by transmission electron microscopy (TEM). Briefly, samples were prepared by dropping one drop of liposome dispersion onto a copper grid coated with a carbon membrane. The samples were dried and visualized under Hitachi H-600 TEM (accelerating voltage of 200 kV). Gd-DTPA concentration in liposomes was determined by inductively coupled plasma mass spectrometry (ICP-MS) (4500 ICP-MS, Hewlett-Packard, DE, USA).
In vitro stability of the liposomes was monitored over a time period of 120 h. Briefly, the liposomes were incubated with PBS or 10% (v/v) FBS in PBS at 37°C over 120 h. An aliquot of liposome solutions was collected to measure the particle size on a Zetasizer Nano S.

The particle size and zeta potential of the nanosuspension were measured using dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern, UK) at 25°C. The size variation trend of nanosuspension kept at 4°C was monitored using DLS over 14 days. The morphology of the nanosuspension was observed using transmission electron microscopy (TEM) (H-600 TEM, Hitachi, Japan) with negative staining using 2% phosphotungstic acid. The ultraviolet-visible (UV-vis) absorbance spectra of free SLB, SLB-M, NS-SLB and NS-SLB-HC dissolved in DI water were obtained within a wavelength range of 200–500 nm using a UV-vis spectrophotometer (UV-2600I, Shimadzu, Japan). The encapsulation efficiency (EE) was studied with sephadex G-50 in separating nanoparticles from nanosuspensions and determined using high performance liquid chromatography (HPLC) (UltiMate^® 3000, Thermo Scientific, USA). While drug loading efficiency (LE) of SLB were determined using HPLC, calculated using the following equation.31 (link)
EE (%, w/w) = W_{entrapped SLB} / W_{fed SLB} × 100%
LE (%, w/w) = W_{loaded SLB} / W_{nanosuspension} × 100%.