Minispin

For serum stability studies (Powell et al., 1993 (link)) 10 μL of an aqueous peptide stock solution (3 mg/mL) was added to 390 μL pooled mouse serum in triplicate. The peptide-serum mixtures were thermostated at 37°C. After 0, 30, 120, and 240 min, 95 μL aliquots were mixed with 25 μL trichloroacetic acid (15%, w/v) and incubated for 10 min on ice. The precipitated proteins were separated by centrifugation (5 min, 12,000 × g, Eppendorf MiniSpin). The supernatant (90 μL) was neutralized with 8.5 μL aqueous sodium hydroxide solution (1 M) and mixed with 3% aqueous acetonitrile containing 0.1% TFA (121.5 μL). After centrifugation (5 min, 12,000 × g, Eppendorf MiniSpin) a 100 μL solution was analyzed by RP-HPLC using a Jupiter C18 column (2.0 mm inner diameter, 150 mm length, 5 μm particle size, 30 nm pore size; Phenomenex, Torrance) on a Beckman Gold HPLC System at 60°C. The gradient from 0.1% TFA in water (eluent A) to 60% acetonitrile in 0.1% aqueous TFA (eluent B) was developed using 1% B/min and the amide bond was monitored at 214 nm.

P. asymbiotica subsp. australis was inoculated from stock cells stored at −80 °C into a standard LB media and were grown in 30 °C under an orbital shaking overnight. Bacterial growth was tracked by measuring the optical density of the broth at 600 nm (OD₆₀₀) using a spectrophotometer (Biochrom Ltd., BioPhotometer WGA CO 8000 Biowave). Cells were harvested when reaching an OD₆₀₀ = 0.5. The culture was centrifuged at 4,300 g and room temperature for 2 minutes (Minispin, Eppendorf, Germany) and cells were then washed three times with PBS buffer. Cells were diluted in PBS to OD600 of 2.0 and kept in a fridge until performing the aggregation assay.

The amount of dArb entrapped in the NLCs was evaluated by an ultrafiltration method using Vivaspin filter tubes (Vivaspin, Goettingen, Germany) with a filter membrane that has a 3 kDa molecular weight cut-off. One millilitre of undiluted dArb-loaded NLCs was placed in the upper chamber of the tube, which was centrifuged at 13,000 rpm for 45 min (Minispin, Eppendorf, Hamburg, Germany). The filtrate was then diluted with methanol, and the amount of un-entrapped dArb was measured using validated high performance liquid chromatography (HPLC) (Waters 1525, Milford, MA, USA) with a reversed C-18 column, methanol:water (60:40) as the mobile phase, and a 280 nm detector. The EE was calculated with the following equation:
$EE(\%)=\frac{\mathit{Initial}\mathit{dArb}-\mathit{Unentrapped}\mathit{dArb}}{\mathit{Initial}\mathit{dArb}}$