Alltech 3300

For extracting the encapsulated siRNA, OGP (Octyl β-D-glucopyranoside; Carbosynth, UK) was used. To determine unentrapped siRNA, specimens of liposomes were centrifuged at 4000 RCF for 45 min, by means of a Vivaspin centrifugal concentrator (MWCO 300 kDa). After extensive vortex, siHSV concentration was determined by the NanoDrop method against a calibration curve, made of empty liposomes (with similar lipids concentration) spiked with different concentrations of siRNA diluted in TE buffer (in duplicates). The actual siRNA loading (µg/mL) and the encapsulation yield (%) were calculated using the following equations: $$\mathrm{siRNA} \mathrm{loading}=\mathrm{Conc}. \mathrm{of} \mathrm{total} \mathrm{siRNA}-\mathrm{Conc}. \mathrm{of} \mathrm{unentrapped} \mathrm{siRNA}$$
$$\mathrm{Encapsulation} \mathrm{yield} (\%)=\left(\frac{\mathrm{siRNA} \mathrm{loading}}{\mathrm{Initial} \mathrm{siRNA} \mathrm{conc}.}\right)\times 100$$
The content of phospholipids in the liposomes was determined by means of HPLC (Waters, Milford, MA, USA) equipped with an ELS detector (Alltech 3300, Grace, Salt Lake City, UT USA), as described previously [46 (link)].

Culture supernatants obtained after centrifugation of the culture were filtered on 0.2 µm MiniUniPrep filter (Whatman). When following “method A”, the filtered supernatants were analyzed on an Atlantis T3 column (250 mm × 4.6 mm, 5 μm, 30 °C) using an Agilent 1200 HPLC instrument equipped with a quaternary pump. Samples were eluted with isocratic 0.1% HCOOH in H₂O (solvent A) / 0.1% HCOOH in CH₃CN (solvent B) (95:5) at 1 ml/min for 5 min followed by a gradient to 100% solvent B over 45 min. Molecules that were present in the supernatant were detected by an Alltech 3300 evaporative light scattering detector (ELSD) (Grace).
When necessary, an analysis of the cell content was made. The mycelium pellet obtained after centrifugation of 2 ml of culture, was washed twice with 2 ml of water, suspended in 400 µl of water and broken down with glass beads using the FastPrep-24 apparatus (MP Biomedicals). A cell-free extract was obtained after centrifugation (13,000 g, 4 °C, 30 min) to remove cell debris.
LC-MS analyses were performed using an Agilent 1100 HPLC coupled via split system to an Esquire HCT ion trap mass spectrometer (Bruker) set in positive and negative modes. Multiple analyses were made. Some of them were made using “method A”, some other using the conditions described by^{15 (link)} for CDPS 1–47 and called “method B”.