Glass vial

Stock solutions of [Cho][DHP], [Cho][OAc], and [Cho][Cl]
were prepared as previously described.^{63 (link)}Table 5 shows the
composition of the formulation buffers developed herein. For each,
stock solutions were prepared. The specified components were dissolved
in ultrapure water (ELGA LabWater, High Wycombe, UK) in a glass vial
(Thermo Fisher Scientific Inc., Waltham, MA, USA) and mixed by means
of vortex and sonication for 30 min at 25 °C.
Ionogel formulations were prepared by stirring each
IL with silk
fibroin solution at 25 °C, and the desired formulation buffer
(Table 5) was added
dropwise to achieve a silk fibroin/formulation buffer/IL ratio of
1:1.6:12.8, by weight. In the absence of IL, systems contained a silk
fibroin/formulation buffer ratio of 1:1.6 by weight and prepared as
before. To produce the PVP inclusive formulations, silk fibroin and
PVP were mixed at a ratio of 1:1 by weight, and the desired formulation
buffer in the presence and absence of IL was added as before. Each
system developed herein consisted of 4 mg/mL of phenobarbital. Sodium
hydroxide and hydrochloric acid were added dropwise to obtain a formulation
pH of 6.5; ensured by pH measurement with the pH electrode Mettler
Toledo InLab Micro (Wolflabs, Pocklington, York, UK).

Newly eclosed flies were collected in groups of 20 and aged for three to five days, after which the heat-induced seizure assay was performed as previously described (Sun et al. 2012 (link)). Briefly, a single fly was put into a 15 × 45 mm glass vial at room temperature (Thermo Fisher Scientific, MA) and allowed to acclimate for two to 10 min. The glass vial was then submerged in a water bath at the specified temperature for two minutes, during which the fly was video-taped and assessed for seizure behavior every five seconds. Seizure behavior was defined as loss of standing posture followed by leg shaking.

Once in the 8 M urea, 10 mM HEPES pH 8 buffer, samples were quantified by BCA assay (Thermo Fisher Scientific #23225). DTT (5 mM final concentration) was added into 50 µg of proteins and samples were boiled for 2 min. After a 30-min incubation at room temperature, chloroacetamide (7.5 mM final concentration) was added to the mixture. Solutions were then incubated in the dark, at room temperature for 20 min. 50 mM of NH₄HCO₃ was added to each tube to reduce the final concentration of urea to 2 M. Peptide digestion was performed by adding 1 µg of trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega Corporation, WI, USA) and incubating each sample overnight at 30°C. The samples were then acidified to a final concentration of 0.2% TFA. For samples from isolated nucleoli, a fraction of the digested sample was collected, at this step, to create the DIA spectral library generation and was processed as described in the next paragraph. On the other hand, remaining samples were cleaned using ZipTips C18 column (EMD Millipore, Burlington, VT), lyophilized in speedvac and finally resuspended in formic acid 1%. Peptides were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific) at a wavelength of 205 nm. The peptides were then transferred into a glass vial (Thermo Fisher Scientific) and keep at −20°C until analysis by mass spectrometry.

Of each freshly prepared sealer (n = 12, 3 per group with the experiment performed in triplicates), 200 μL was placed at the base of individual wells of a sterile 12-well flat bottom plate (Star Lab, Milton Keynes, UK) using a 1-mL sterile syringe. 10 mL of HBSS (Gibco®, Grand Island, NY, USA) was immediately added to each well and stored at 37 °C. The HBSS was replaced at each endpoint (15, 30, 60, 120 min and 1, 3, 7, 14, 28 days). The collected HBSS solution was transferred to a glass vial (Fisherbrand, Loughborough, UK) and vortexed for one minute. Determination of the HBSS solution pH was performed at room temperature using a pH meter (FiveEasy Cond Meter F30, Mettler Toledo, Greifensee, Switzerland) and a temperature compensated pH electrode (pH electrode InLab Easy, Mettler Toledo, Greifensee, Switzerland) previously calibrated at five points (pH 2.00, pH 4.01, pH 7.00, pH 9.21, pH 12.00) using buffer solutions (Technical Buffer Solutions, Mettler Toledo, Greifensee, Switzerland). The mean and standard deviation of each were calculated.

97.5 mol% DOPG, 1.5 mol% DOPE-PEG2k and 1.0 mol% DOPE-Rh (or DOPE-NBD) were mixed in a glass vial (50 × 12 mm, 1481–1582, Fisher Scientific) to form a mixture containing 2.5 mg of lipids. For unPEGylated liposomes, 99 mol% DOPG and 1 mol% DOPE-Rh were used. Chloroform in the mixture was evaporated using a nitrogen stream to form a thin lipid film within the vial, then the vial was put in a vacuum desiccator for at least 2 h to further remove residual chloroform. The anhydrous lipid film was hydrated with 1 mL PEG-rich phase to produce a solution with a lipid concentration of 2.5 mg/mL. This solution was firstly processed with five free-thaw cycles and then extruded with 11 passes through a 0.1 µm filter (Whatman filters, Avanti Mini-Extruder) to produce liposomes. The liposomes were characterised via transmission electron microscopy (JEM-2100F (JEOL, Ltd) fitted with an Orius SC1000 CCD Camera (Gatan, Inc.) and dynamic light scattering (Zetasizer Ultra, Malvern Panalytical), respectively (Supplementary Fig. 9).