Rotary evaporator

The B. carterii Birdw oleoresin samples used for this study was provided by United Kingdom Essential oils and authenticated by Steven Holmes as a part of industrial procurement quality control. Voucher specimens are stored at The University of Salford, United Kingdom.
B. carterii oleoresin was macerated to a fine powder and then extracted using either acetonitrile (99.5%) (AcN), distilled water (dH₂O), ethanol (99%) (EtOH), methanol (99.5%) (MeOH) or propan-2-ol (99.5%) (PrOH) at a final concentration of 100 mg/mL. The mixture was then stirred continuously for 2 h at room temperature. After 2 h, the supernatant was removed, and fresh extraction solvent added. This process was repeated three times. The supernatants were pooled then filtered through Whatman no 1 filter paper, before being dried to a powder using a rotary evaporator (Eppendorf, United States) and stored at −20°C. All extracts used for further experimentation were dissolved in dimethyl sulphoxide (DMSO) to a stock concentration of 50 mg/mL.
Each of the extracts is characterized by gradient high-performance liquid chromatography with diode-array detection (HPLC-DAD) as described in Supplementary Data S1.1 with spectra for each extraction method shown in Supplementary Figures S1–S4.

DCPIB (Tocris), NPPB (Tocris), FFA (Sigma), Cytochalasin D (Sigma) and Calcein‐AM (Biolegend) were obtained as powders dissolved in DMSO to ×200 concentrated stocks and stored at −20°C. pHrodo‐SE (ThermoFisher) was dissolved in DMSO to 10 mg/ml and stored in aliquots at −80°C. Human Aβ_1‐42 (Eurogentec) was dissolved in hexafluoro‐2‐propanol, dried into films under a rotary evaporator (Eppendorf) and stored at −80°C. Tomato lectin conjugated to DyLight 594 (Vector Biolabs) was washed twice through a centrifugal filter (10 kDa MWCO) to remove sodium azide, sterile filtered and stored as a 1 mg/ml stock at 4°C.

In a dry 25 mL rotary evaporation bottle, 10 mg DPPC and 4 mg DSPE-PEG-Biotin (2000) were mixed and 2 mL chloroform was added to dissolve the mixture. Then, 200 μL NIRF agent IR783 liquor (dissolved in chloroform, 1 mg/mL) was added. In a rotary evaporator (New Brunswick Scientific, New Brunswick, NJ), rotary evaporation was conducted at 120 rpm and 55°C. After 10 minutes, chloroform was completely evaporated and a uniform light green phospholipid thin film was developed. Subsequently, 1.5 mL hydration liquid (10% glycerol and 90% 1x PBS, v/v) was used for the film hydration. The bottle was put into an incubator-shaker (New Brunswick Scientific, NJ) at 130 rpm under 37°C for 60 min, then the suspension of IR783-loaded liposomal film was prepared. The suspension was transferred into a tube. After the air in the tube was removed and C₃F₈ gas was inflated, the tube was placed in a mechanical oscillator (Ag and Hg mixer, Xi'an, China) for 90 s to produce bubbles. To combine biotinylated anti-ErbB2 Affibody molecules with IR783-loaded NBs, the avidin-biotin method was applied. To prevent fluorescence quenching, tin foil was used to cover all bottles and tubes. In the control group, we used SonoVue. Finally, the IR783-NBs-Affibody was sterilized by CO60 irradiation for 30 min.

Fixed-ratio mixtures of the phospholipids DSPE-PEG (2000) and DPPC (7, 14, 21 or 28 mg) were added to 25-ml rotary evaporation bottles and dissolved in 2 ml of chloroform. A small amount of the fluorescent membrane probe DiI (red fluorescence) was then added. Rotary evaporation was performed for 10 min at 55 °C and 120 rpm/min in a rotary evaporator (New Brunswick Scientific, Enfield, CT, USA). After the chloroform evaporation, milky white phospholipid thin films were observed on the rotary evaporation bottle walls. The milky white phospholipid thin films were hydrated with 0.5, 1, 1.5 or 2 ml of hydration liquid consisting of 10% glycerol and 90% 1 × PBS (V/V). The rotary evaporation bottles were placed in an incubator-shaker (New Brunswick Scientific) at 37 °C and 130 rpm for 60 min. Then, 500 μl of each suspension were transferred into four vials sealed with plastic caps. The air in the vials was replaced with C₃F₈ gas using a 50-ml syringe with a long, fine needle. Finally, every vial was oscillated for 45 s in a mechanical oscillator (Ag and Hg mixer, Xi’an, China) to generate the bubbles. The bubbles in each vial were separately diluted to 8 ml in PBS. All bottles and vials were covered with aluminum foil to prevent fluorescence quenching. In addition, commercial SonoVue microbubbles were used as the control.