Sonifier 450

Liposome complexes consisting of TM4SF5 B cell epitope and CpG-DNA (MB-ODN 4531(O)) co-encapsulated with DOPE: CHEMS were prepared as reported previously (12) (link). Briefly, DOPE and CHEMS were mixed in 10% ethanol at a molar ratio of 1：1, evaporated with nitrogen gas to make a solvent-free lipid film, and resuspended in a mixture containing equal volumes of water-soluble MB-ODN 4531(O) (50 μg) and peptide (50 μg), followed by vigorous stirring at room temperature for 30 min. After adjusting the pH to 7.0, the complex of peptide and Lipoplex(O) was sonicated lightly for 30 s with a sonicator (Sonifier 450, Branson Ultrasonics). After the complex was filtered with a 0.22 μm filter, it was freeze-thawed three times with liquid nitrogen.

The drug extraction procedure was modified from those previously described⁶ (link). Liver, spleen, kidney, and brain were weighted for wet weight and then cut into small pieces. The tissue homogenates were prepared by adding pre-cooled normal saline to tissue at a ratio of 5:1 (v/w) and then separately by a homogenizer (Sonifier 450; Branson Ultrasonics, Branson, MS, USA). Then the obtained homogenates were stored at −80 °C until analysis.
An internal standard method was adopted to determine the concentration of TDE in the tissue samples. An aliquot of 200 μL of tissue homogenate was mixed with 50 μL of internal standard solution (corydaline; CDE, 5 mg/L) in a glass tube and vortex-mixed for 30 s. Then one mL n-hexane-2/propanol (95/5) was added in the glass tube, and then vortex-mixed for 3 min; the obtained mixture was centrifuged at 16,000 × g for 15 min. The upper organic layer was transferred to a clean glass centrifuge tube and dried under a stream of nitrogen at 40 °C. The dried samples were reconstituted with 100 μL mobile phase, shocked in vortex for 1 min, and then centrifuged for 5 min at 16,000 × g at 4 °C. After centrifugation, 100 μL supernatant was kept at −80 °C before HPLC analysis.

The suspensions were tip-sonicated in batches of 200 mL for 3 min (2–1 s on–off cycles) at an amplitude of 30%. A 2 cm diameter probe (Sonifier 450, Branson Ultrasonics Co.) was used, and an ice bath helped to avoid overheating of the sample. The concentration of the suspension was adjusted by evaporation at 50 °C inside a Teflon container with gentle magnetic stirring until a concentration of ca. 2.5 wt% was attained. A new tip-sonication step was performed for 2 min at 40% amplitude (2–1 s on–off cycles). Centrifugation at 9000 rpm for 10 min (with an additional 4 min of ramp up and 4 min ramp down) was carried out and the supernatant was kept, eliminating large particle aggregates possibly generated during the evaporation step. The concentration of the resulting suspension was finally adjusted to 2 wt% with Milli-Q water and the final pH was ca. 3. The final ChNC samples were stored at 4 °C and were observed to be colloidally stable over several months.