Silent crusher

Isolation was performed as previously described (Hocker et al. 2013) with modifications. Tissue samples were frozen in liquid nitrogen and stored at −80°C until use. A quantity of 5–20 mg of tissue was weighed and homogenized in 700 μL Qiazol (Qiagen, Germantown, MD) on ice using a Heidolph Silent Crusher (Schwabach, Germany). Nucleic acid extraction was performed using 140 μL of chloroform, the resulting aqueous layer was precipitated in 0.5 mL of isopropanol, and the resulting pellet was rinsed in ethanol, and resuspended in RNase‐free water. RNA quantification was performed using a Qubit fluorometer (Qiagen) and RNA quality was determined using a Fragment Analyzer (Advanced Analytical, Ames, IA). We set a RNA Quality Number (RQN) value cutoff of 5.7 for each sample.

Naringenin nanoparticles were synthesised according to Sahadan et al. (2019). A total of 200 μg of naringenin (Alfabiotech, UK) was first dissolved in 5 mL of methanol (Thermo Fisher, USA), followed by the addition of 1 g of Pluronic F127 (Sigma Aldrich, USA). The mixture was homogenised by using a Silent Crusher (Heidolph, Germany) at 10,000 rpm for 5 min in an ice bath. Next, 50 mL of 2% PVA solution (R&M Chemicals) was added into the mixture and agitated at the same agitation speed for 10 min. The transparent solution was kept at −80°C and followed by a freeze-drying process (Labconco, USA). The freeze-dried nanoparticles were kept in a desiccator at room temperature (25 ± 2°C) prior to use. The blank nanoparticles that served as a negative control for antimicrobial assays were synthesised by replacing the naringenin solution with methanol. The nanoparticle powder was dissolved in 30% Tween 20 solution to an appropriate concentration, prior to filtration process via 0.22 μm pore size filter (Millipore, USA).

AT-NLCs were prepared using a high-shear homogenization method associated with sonication. First, the lipid phase was prepared by heating solid lipids (Stearic acid or Gelucire 43/01 or their combinations) at 10 °C above their melting points by a heating magnetic stirrer (Brandstead/Thermolyne, Swedesboro, NJ, USA). The liquid lipid (Labrasol or oleic acid or their combinations) was added to the solid lipid. AT (with or without lecithin) was added to the lipid phase. The aqueous phase, which contained surfactant (Tween 80 or Poloxamer 188) and bidistilled water, was heated at the same temperature of the lipid phase separately. After reaching the same temperature, the heated aqueous phase was added dropwise to the molten lipid phase and mixed at 600 rpm for 10 min by the heating magnetic stirrer. Subsequently, the mixtures were homogenized using a Heidolph Silent Crusher^® homogenizer (Heidolph, Schwabach, Germany) for 10 min. Finally, the formulations were subjected to a digital sonifier (Branson, Danbury, CT, USA) for 5–10 min [23 (link)]. All formulations were kept in tightly closed containers for further investigations.

The aqueous phase containing 10 wt% or 15 wt% of lyophilized microorganisms was firstly prepared in DDW. Then, the MCT oil phase (2.5 g) was added to the aqueous phase (2.5 g) and magnetically stirred (700 rpm for 2 min). Homogenization (16,000 rpm for 10 min) was carried out using a high speed homogenizer (Heidolph Silent Crusher, Schwabach, Germany) equipped with type 8F stainless steel probe.

Thymol nanoparticles were synthesized using polyvinyl alcohol (PVA) as encapsulant (16 (link)). Firstly, 0.3 g of thymol (Solarbio, Beijing, PR China) was mixed in 5 mL of 25 % ethanol (Thermo Fisher, Waltham, MA, USA). Next, 50 mL of 2 % Pluronic F127 (Sigma-Aldrich, Merck, St. Louis, MO, USA) were mixed with thymol solution using silent crusher (Heidolph, Schwabach, Germany). Then, 50 mL of 2 % PVA solution (Sigma-Aldrich, Merck) were added and mixed at 8944×g for 5 min until a clear solution was observed. Then, the solution was kept in a freezer (-80 °C), prior to freeze-drying (Labconco Freeze Dry System, Missouri, MO, USA). The freeze-dried nanoparticles were kept in a desiccator prior to use, and these particles are called Thy/PVA nanoparticles in this study. A control was provided by replacing the thymol solution with ethanol and these particles are called blank nanoparticles. For antimicrobial assays, the nanoparticle was dissolved in 20 % Tween 20 to a desired concentration and strained through a filter (0.22 μm pore size; Millipore Sigma, Billerica, MA, USA) prior to use.

Weighed amounts of DDA and MMG-1 were dissolved in 99% (v/v) EtOH and mixed in a glass vial at an 82:18 M ratio. The lipid mixture was dried under a gentle N₂ stream for 2 h followed by air-drying overnight to remove trace amounts of solvent. The lipid film was rehydrated in Tris-buffer (10 mM, pH 7.4) by HSM by using a Heidolph Silent Crusher equipped with a 6F shearing tool at 60°C and 26,000 rpm for 15 min. Subsequently, poly(I:C) was added slowly to the liposomes with concomitant HSM at 60°C. The final concentrations in the resulting dispersion were 2.5/0.5/0.5 mg/ml DDA/MMG-1/poly(I:C). Fluorescently labeled CAF09 used for in vivo tracking studies was prepared by addition of DiO dissolved in EtOH during the preparation of the lipid film, resulting in a concentration of 0.03 mg/ml in the final formulation.