Vivaspin 6

Phase-change materials (PCM) NPs were fabricated following a previously reported method with minor revisions 36 . Briefly, lauric acid and stearic acid (4:1 by weight) were dissolved in methanol at 4 mg/mL. In order to enhance the stability of NPs and the dispersion in biological fluids, the hydrophilic polymeric shell with antibiofouling properties was modified onto the surface of PCM NPs. Lecithin and DSPE-PEG5000 (3:1 by weight) were dissolved in 4% aqueous ethanol at 1 mg/mL. Phospholipid solution (3 mL) was heated to 50 °C for 30 min. The PCM solution (600 μL) mixed with the desired payloads (48 μL 2.5 mg/mL DIR or Cy5.5 in DMSO and/or 48 μL 2.5 mg/mL CuET in DMSO) was then added dropwise into the preheated phospholipid solution, followed by vigorous vortex for 3 min. After cooling down in the ice water for 2 min, the cloudy solution was warmed up to ambient temperature in 2 h, and was then vortexed for 2 min, followed by filtration through a 0.2 µm surfactant-free cellulose acetate membrane (Thermo Fisher Scientific). The unencapsulated molecules and organic solvents were removed using a VIVASPIN 6 centrifugal concentrator (Sartorius, MWCO=10 kDa). After washing with water for 3 times, the resultant nanoparticles were suspended in water for further use.

To express the cell attachment proteins MRV σ1 and NBV σC in mammalian cells, 293T cells were transfected with p3×FLAG-T3D-σ1 or p3×FLAG-MB-σC using 1 mg/ml polyethyleneimine solution (Cosmo Bio). After 48 h of incubation, the cells were collected and lysed in buffer containing of 50 mM Tris–HCl pH 7.4, 150 mM NaCl, and 1% Triton X-100. The recombinant proteins were purified from the soluble fraction using ANTI-FLAG M2 Affinity Gel (Sigma) according to the manufacturer’s instructions. The purified proteins were competitively eluted using 3 × FLAG peptide (Sigma). The proteins were dialyzed using Vivaspin 6 (Sartorius) and used for cell-surface binding assays.

Expression and isolation of crude membranes were performed as described above. For solubilization, 2% (w/v) DDM and 0.2% (w/v) CHAPS (anagrade, Affymetrix) were added to gently thawed membranes, and the sample was stirred for 1 h at 4 °C. The solubilizate was ultracentrifuged (60,000 × g for 1 h) to discard non-solubilized material and protein aggregates. The supernatant was incubated for 30 min with GPF-nanobody-coupled agarose beads (GFP-Trap_A, Chromotek) at 4 °C. Then the flow-through was discarded, and the beads were washed with 10 column volumes of buffer A, supplemented with 0.2% (w/v) DDM and 0.2% (w/v) CHAPS. To elute hTRPV3, the fusion protein was cleaved off overnight by incubation with PreScission protease (0.3 mg·ml⁻¹) at 4 °C, leaving the GFP-His tag bound to the beads. The total elution (eluate + 5 column volumes of wash) was then concentrated to ∼1 ml (100 kDa cut-off, Vivaspin 20, Sartorius) and loaded on a Superose 6 10/300 GL gel filtration column (AKTA purifier system, GE Healthcare). The running buffer consisted of buffer B (100 mm NaCl, 10 mm Tris, pH 7.5), supplemented with 0.03% (w/v) DDM and 0.1% (w/v) CHAPS. After analysis on SDS-PAGE, the peak fractions were compiled and concentrated (100 kDa cut-off, Vivaspin 6, Sartorius).

Exosomes were labeled with 4 μg/ml PKH26 dye (Sigma, USA) for 5 min, and then 3% BSA (Aladdin, China) was added to stop the staining reaction. After centrifugation using a 300 kDa ultrafiltration centrifuge tube (Sartorius Vivaspin 6, Germany), exosomes were washed three times with PBS to allow removal of unbound dye. Labeled exosomes were added to MLE-12 cells for 48 h, and the images were observed under a fluorescence microscope (control group added unlabeled exosomes). For a shuttling assay of a Cy3-labeled-miRNA precursor, MenSCs were transfected with Cy3-labeled Let-7 mimic (Guangzhou RiboBio, China). Then, exosomes secreted from MenSCs transfected with Cy3-labeled Let-7 mimic, or Cy3 alone was extracted and added to MLE-12 cells (not expressing Cy3) for 48 h. The fluorescence intensity of Cy3 in the alveolar epithelial cells was observed by fluorescence microscopy.

Three ZIKV strains were used, each representing a viral lineage: DAK 84 (Senegal 1984, African genotype), MRS_OPY_Martinique_PaRi_2015 (Martinique 2015, Asian genotype, American lineage) and MASS 66 (Malaysia 1966, Asian genotype, Asian lineage). Viruses were provided by the Emergence Virus Unit (Marseilles, France) via the European EVAg project. Lyophilizates were resuspended in sterile distilled water and inoculated onto Vero cells (ATCC, ref. CCL-81) for viral production using a multiplicity of infection of 0.1 and DMEM medium supplemented with 2% fetal bovine serum (FBS). Supernatants were collected after three days of growth and stored at −80 °C prior to vector competence experiments. The viral titer of each virus strain was determined by serial 10-fold dilutions of viral stock on Vero cells, and was expressed as 50% tissue culture infective dose per milliliter (TCID₅₀/mL). In the case of MRS_OPY_Martinique_PaRi_2015 ZIKV strain, virus stock was centrifuged using Vivaspin 6 centrifugal concentrator (Sartorius, Stonehouse, UK) to achieve the final concentration at 10⁷ TCID₅₀/mL used in the oral challenge experiments.

One aliquot of the soluble extract of WB was used to fractionate it by MW using Vivaspin 6 centrifugal concentrators (Sartorious) with a cut-off size of 300,000-Da. The upper part of the tube was filled with the soluble extract and centrifuged (3,000×g, 3.5 h, 4°C). After centrifugation, the upper part was adjusted with DEMI water to the same volume retrieved in the bottom container to achieve the same sample-volume. Two fractions were obtained: 1) >300-kDa and 2) <300-kDa. Finally, these fractions were immediately freeze-dried and kept at RT until further testing.

For determination of TAN EE %, samples of different NLCs formulations were properly diluted with water and then subjected to centrifugation using Vivaspin^® 6 centrifugal ultrafilters (MWCO = 100,000, Sartorius, USA) at 6000 rpm for 30 min at 4 °C. The concentration of unentrapped TAN in the filtrate was quantified spectrophotometrically at 270 nm [8 (link)]. EE % was calculated using the following equation: $$\%EE=\frac{Totaldrugamount\left(m,g\right)-amountofunencapsulateddrug(mg)}{Totaldrugamount(mg)}\times 100$$