D trehalose dihydrate

Primary Mouse Embryonic Fibroblasts (MEFs) were prepared from CD-1 mice embryos of 17–18 days of gestation following standard procedure. The mice were obtained from the animal house at the Instituto de Fisiología Celular, UNAM, following IACUC guidelines (approved protocol CICUAL SCO51-18). Cells were grown in DMEM (10,569, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (16,000, Gibco) and penicillin/streptomycin 100 U/mL (15,140, Gibco) in the presence of 5% of CO₂ at 37 °C. The cells were incubated for 6 h with IP-1 peptide at different concentrations (0, 20, 30, 40 and 50 μM) prepared in DMEM. HEK2913T cells were seeded in 12-well plates (1 × 105 HEK293T cells per well) in 1 mL of supplemented DMEM (Invitrogen, Grand Island, NY, USA).
Iztli peptide 1 (IP-1, KFLNRFWHWLQLKPGQPMY) was synthesized and purified by Anaspec, Inc. (Fremont CA 94555, USA). The disaccharide D-(+)-Trehalose dihydrate (T0167-100G SIGMA-ALDRICH, St. Louis, MO, USA) was used as a positive control of autophagy induction at 100 mM for 5 h. Chloroquine was used at a final concentration of 40 µM for thirty minutes before treatment with IP-1 or trehalose. All reagents were diluted in water and used at indicated concentration in each experiment.

MEL (4-hydroxy-2-methyl-n-(5-methyl-2-thiazolyl)-2 H-benzothiazine-3-carboxamide-1,1-dioxide) was applied as model drug and acquired from EGIS Plc. (Budapest, Hungary). Soluplus^® (BASF GmbH, Hanover, Germany) was used as micelle-forming agent. Ethanol (Merck, Ltd., Budapest, Hungary) as organic solvent was used during our experiment. Microcrystalline sodium hydroxide (NaOH) as formulation excipient, chemicals for Simulated Nasal Electrolyte Solution (SNES) [16 (link)] which combined 8.77 g sodium chloride (NaCl), 2.98 g potassium chloride (KCl), 0.59 g and anhydrous calcium chloride (CaCl₂) in 1000 mL of deionized water at pH 5.6 as well as disodium phosphate (Na₂HPO₄), monopotassium phosphate (KH₂PO₄) for pH 7.4 Phosphate-buffered saline (PBS) dissolution media and the cryoprotectant d-trehalose dihydrate were acquired from Sigma-Aldrich Co., Ltd. (Budapest, Hungary). Purified water for the experiments was filtered using the Millipore Milli-Q^® (Merck, Ltd., Budapest, Hungary) 140 Gradient Water Purification System.

d-(+)-Trehalose dihydrate, d-mannitol, and dextran (M_r 40,000 kDa) were purchased from Sigma-Aldrich (Ontario, Canada). Recombinant Vesicular Stomatitis Virus-based vaccine vectors (VSVAg85A and VSVGFP) were produced in the vector facility of McMaster Immunology Research Centre as described previously^{1 (link)}. The vaccine vector is replication-competent containing the VSV wild-type glycoprotein.

For the final nanoparticle dispersion, an endotoxin quantification assay was performed with an Endosafe Nexgen-PTS device. No endotoxin was detected in all synthesized nanoparticles (<0.1 EU/mL). To calculate the number of nanoparticles per volume unit, the Ca2⁺ concentration was measured by AAS and then tentatively expressed as the most common calcium phosphate, i.e., hydroxyapatite, Ca₁₀(PO4)₆(OH)₂. The nanoparticle concentration in 1 mL dispersion is calculated from the measured calcium phosphate concentration and the density of hydroxyapatite (3140 kg/m³) assuming a complete spherical morphology. Additionally, the number of Env units on the nanoparticle surface was determined by UV-Vis spectroscopy with an AlexaFluor®-488 labelled Env protein and UV microvolume spectroscopy (“Nanodrop”) (see [22 (link)] for typical calculation steps to obtain these data). For storage and transportation, the nanoparticle dispersion was lyophilized according to our previously reported protocol [23 (link)]. 20 mg D-(+)-trehalose dihydrate (Sigma-Aldrich Corp., St. Louis, MO, USA) were added to 1 mL of the nanoparticle dispersions as cryoprotectant followed by shock-freezing with liquid nitrogen and lyophilization for 72 h at 0.31 mBar and −10 °C. Immediately before the application, the nanoparticles were redispersed in 1 mL ultrapure water and gently sonicated with an ultrasonication bath.

Bioneedles (15 mm long and 1 mm wide, internal volume of 5 μl) were obtained from the Bioneedles Technologies Group. Influenza A/PR/8/34 whole inactivated virus was produced by Intravacc. The process was based on egg virus propagation and β-propiolactone virus inactivation [12] (link). Split and subunit vaccines were produced by solubilization of WIV with n-octyl-β-D-glucopyranoside (Sigma-Aldrich) as described previously [8] (link). Virosome vaccine was produced as described previously [13] (link). All vaccines were concentrated with Centriprep centrifugal filters (Millipore) with a molecular weight cut-off (MWCO) of 10 kDa, and formulated in HBS (20 mM HEPES, 125 mM NaCl, 9 mM CaCl₂, 5 mM MgCl₂). Vaccine formulations for bioneedles contained 2.5% (w/w) D-trehalose dihydrate (Sigma-Aldrich) as a stabilizer. Influenza vaccine containing bioneedles (subunit, split, virosomal and WIV vaccine) were prepared by filling bioneedles with 5 μL of 1 mg/mL (HA content) liquid vaccines from the hollow back of the bioneedle using specially designed filling apparatus, and frozen on a metal plate at minus 50°C [11] (link). Next, bioneedles were freeze-dried using a Zirbus Sublimator 3×4×7 (Zirbus Technology). Lyophilized bioneedles were stored in glass vials with rubber stoppers under ambient air and relative humidity.

Trivalent IPV, containing the inactivated Mahoney strain for type 1, MEF for type 2 and Saukett for type 3, was obtained from the process development department of Intravacc (Bilthoven, The Netherlands). The ten times concentrated trivalent bulk used in this study was determined at a nominal concentration of 400-80-320 DU/ml by ELISA as described (24 (link)).
The excipients sucrose, D-sorbitol, D-trehalose dihydrate, mannitol, L-glutamic monosodium salt monohydrate (MSG), glycine, myo-inositol, magnesiumchloride hexahydrate, lithium chloride and ovalbumin were all purchased from Sigma (St. Louis, MO). Peptone (vegetable) and dextran (6 kDa, from Leuconostoc ssp.) were from Fluka (Buchs, Switzerland) and sodium chloride was from Merck (Darmstadt, Germany). To prepare 10 mM McIlvaine buffer, 10 mM citric acid (Sigma-Aldrich, St. Louis, MO) was added to 10 mM disodium hydrogen phosphate dehydrate (Na₂HPO₄) (Fluka, Buchs, Switzerland) in a ratio of 1:6 resulting in a pH-value of 7.0. All excipients used were of reagent quality or higher grade.