H 7000fa

The prepared MVs were pipetted onto Formvar carbon-coated copper grids. Then, the adsorbed MVs were negatively stained with 2% (w/v) phosphotungstic acid (PTA, pH 6.8) for two minutes. Next, the grids were washed with distilled water and then air dried. The MVs were observed using a transmission electron microscope (Wuhan Institute of Virology, 100 kV Hitachi H-7000FA).

hADSCs at passage 3 were cultured under hypoxia (5% oxygen) and normoxia (20% oxygen) until cells reached 70–80% confluency. The medium was then replaced with serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 for 24 h to collect conditioned medium. Cell debris and apoptotic bodies were discarded after sequential centrifugations at 500 ×g, 3,000 ×g and 10,000 ×g for 5, 15 and 60 min, respectively. The supernatant was then ultra-centrifuged at 100,000 ×g at 4 °C for 1 h using a 45 Ti rotor (Beckman Coulter, Optimal L-80XP, USA). The remaining precipitate was resuspended in PBS and filtered with a 0.22-mm filter (Millipore, Billerica, MA, USA), then ultra-centrifuged at 100,000 ×g at 4 °C for 1 h. EVs were stored at −80 °C until further use. EVs obtained from both conditions were characterised using 80 kV TEM (HITACHI H-7000FA, Japan). The particle size distribution and concentrations of EVs were analysed by ZetaVIEW (Particle Metrix, ZetaVIEW S/N 17–315, Germany) and Nanoparticle Tracking Analysis software (ZetaVIEW 8.04.02). Antibodies against CD9, CD63, Alix, β-actin and calnexin (Abcam, London, UK) were used in western blotting. In addition, the supernatant of EV lysates was prepared, and the protein content was evaluated using a BCA Protein Assay Kit (Sigma, Silicon Valley, USA).

The magnetic nanoparticles were imaged by transmission electron microscopy (TEM) (H-7000FA, HITACHI, Tokyo, Japan) with 110 kV to characterize their core size.

After exposure to β-Thujaplicin for 24 h, cells were harvested and fixed with 2.5% glutaraldehyde for 2 h at 4 °C. Then, cells were washed three times with PBS and fixed in 1% aqueous osmium at 4 °C for 1 h, dehydrated with increasing concentrations of acetone, and embedded in araldite. The ultrathin sections were produced with a microtome (Leica, Jena, Germany) and stained with 3% aqueous uranyl acetate and lead citrate. The results were observed by a transmission electron microscope (Hitachi H-7000FA, Tokyo, Japan) and demonstrated as the number of autophagosomes per cell. Autophagosomes are defined as a double-layer membrane structure containing cytoplasmic contents (mitochondria, damaged organelles, etc.) waiting to be degraded. Images of five view fields were taken for data analysis.

The purified goose red blood cells (1 × 10⁶ cells/ml) were incubated with latex beads (0. 8-μm diameter) at a ratio of 1:10. After incubation at 37°C for 1 h, 2 h, and 4 h, the beads were washed three times with 0.9% sterile buffered saline and fixed in 2.5% glutaraldehyde at 4°C for at least 2 h. Finally, scanning electron microscopy (SEM) (Hitachi SU8010, Japan) and transmission electron microscopy (TEM) images (Hitachi H-7000FA, Japan) were used to observe the phagocytic activity of goose RBC. Scanning electron microscope magnification (× 8.00 k and × 20.0 k) and transmission electron microscopy magnification (× 4.0 k).

The fermentation liquid of the B. thuringiensis engineering strain ACE-38 [15 (link)], which was constructed by transforming the insecticidal gene into the strain BMB171, was stored at room temperature for 1 year and used for phage isolation. The fermentation liquid was first centrifuged at 12,000× g for 10 min and subsequently filtered through a 0.22-μm filter (Millipore, Burlington, MA, USA) to remove the spores and fermentation materials, and then the filtered supernatant was used for phage isolation as previously described using the double-layer overlay method [16 (link)]. The phage propagation, the efficiency-of-plating (EOP) test, and the host range determination were all performed by double-layer overlay assay. The storage stability of the phage was also determined by testing the EOP of the phage suspension after storage for different time periods.
To observe the morphology of the phage virion, the phage was purified by using sucrose density gradient centrifugation as previously described [17 (link)]. The purified phage suspension was deposited onto cuprum grids with carbon-coated Formvar film, strained with 2% potassium phosphotungstate (pH 7.2), and further air-dried. The sample was observed using a transmission electron microscope (TEM, H-7000FA; Hitachi, Tokyo, Japan) at an acceleration voltage of 100 kV.

2-BP/CPT-PLNs were prepared by solvent evaporation and ultrasonic emulsification method as described previously [32 (link)]. Typically, CPT-ss-PAEEP₁₀, 2-BP, and DSPE-PEG were co-dissolved in methanol-chloroform mixed solvent and then dropwise added to deionized water under stirring. The above mixture was evaporated under heating and stirring overnight, then sonicated under an ice-bath for 2 min. The collected suspension was washed with deionized water by ultrafiltration (Millipore, 10 kDa) to obtain 2-BP/CPT-PLNs after removing the residual organic solvent.
The particle size, polydispersity index (PDI), and zeta potential of 2-BP/CPT-PLNs were measured by dynamic light scattering (DLS) technology (Zetasizer Nano ZS90, Malvern). The size and morphology of nanoparticles were characterized by transmission electron microscopy (TEM, H-7000FA, Hitachi). The encapsulation efficacy of CPT-ss-PAEEP₁₀ and 2-BP within 2-BP/CPT-PLNs nanoparticles was measured by fluorescence spectrometer (F-4500, Hitachi) and high-performance liquid chromatography (HPLC, Agilent LC1100, USA), respectively. The assembly behavior, stability, and CPT release behavior of 2-BP/CPT-PLNs were also evaluated by the DLS technology or fluorescence spectrometer.