Snakeskin dialysis tube

The protein was applied to a d-galactose-sepharose affinity column (Thermo Scientific) and eluted using 300 mM galactose in PBS. The fractions were concentrated using Vivaspin 20 ml concentrator tubes (5000 MWCO, PES membrane, Sartorius), and subjected to size-exclusion chromatography on a Superdex75 column mounted on an Äkta FPLC machine, pre-equilibrated with a Tris running buffer (20 mM Tris, 200 mM NaCl at pH 7.5). Fractions with toxin were dialyzed against the Tris running buffer in a Snakeskin dialysis tube (3500 MWCO, Thermo Scientific), concentrated using concentrator tubes to 3–10 mg/ml, and stored at −80 °C.

The Ig fractions from NHP and from the EDTA plasma of patients P24, P25, P26, and P27 (Table 1) were isolated by affinity purification using NAb™ protein A/G 5 mL spin columns (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. In short, 0.85–1.0 mL of EDTA plasma was diluted with binding buffer (0.1 M phosphate, 0.15 M sodium chloride, and pH 7.2) to 10 mL and loaded on the columns. Unbound fractions were washed with binding buffer, and thereafter bound Igs were eluted using 0.1 M glycine, pH 2.5 followed by immediate neutralization with 1/10 volume 1 M Tris, pH 8.5. Subsequently, Ig fractions were dialyzed against phosphate buffered saline using SnakeSkin^® dialysis tubes (Thermo Fisher Scientific, Waltham, MA, USA) with a 10 kD molecular weight cutoff and concentrated to the initial plasma sample volume using Amicon^® Ultra-15 centrifugal filter devices (Merck Millipore, Billerica, MA, USA) with similar cutoff. Total protein concentrations in these samples were measured using NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and varied between 4.73 and 12.48 mg/mL, which are within the normal range for Igs.

The crude enzyme extract (600 mL) was subjected to stepwise ammonium sulfate precipitations from 30 to 90% saturations at 4 °C. The precipitates were harvested by centrifugation at 8000× g for 20 min. The pellet was dissolved in 5 mL of 0.1 mM phosphate buffer, pH 7.0 and dialyzed (10,000 MWCO SnakeSkin^® dialysis tubes, Thermo Scientific, Waltham, MA, USA) prior use. Each fraction was subjected to lipase and protein (Bradford reagent, Amresco, Solon, OH, USA) assays followed by SDS-PAGE.

Cyclodextrin species, namely αCD and βCD, were conjugated to branched PEI of molecular weight (M_n) 25 kDa by means of stepwise 1,1′-carbonyldiimidazole (CDI)-mediated reactions (Scheme 1). Briefly, a DMSO solution containing βCD activated with CDI was added to the aqueous solution of PEI 25k, and after the completion of the reaction, the polymeric material was purified by means of dialysis against water (3.5k MWCO membrane was used). The as-obtained polymeric material (PEI-βCD) was subjected to the reaction with αCD activated with CDI. Finally, after dialysis against water (3.5K MWCO Snake-Skin^® Dialysis Tubes, Thermo Fisher Scientific, Warsaw, Poland) the desired polymeric vector (PEI-βCD-αCD) was obtained. The lyophilization was performed using FreeZone 1 L Laboratory Lyophilizer (LABCONCO, Gadńsk, Poland). The details of synthesis of individual components of the nanoconjugate are given below.

Electrophoretic mobility (µ_e) was determined using a Malvern Nano ZS analyzer (Worcestershire, UK). The measurements for pure BSA and BSA-5FU complexes were made in water with a constant BSA concentration of 1 mg/mL. Measurements were carried out for complexes with BSA/5FU molar ratios of 1:40 and 1:60. Complexes for electrophoretic mobility measurements were mixed for one h and then dialyzed using SnakeSkin™ dialysis tubes (MWCO 3.5 kDa) (Thermo Fisher, Waltham, MA, USA). Dialysis was conducted in deionized water for 24 hours in darkness. All electrophoretic mobility measurements were conducted in the pH range of 2.0–11.0. The pH of solutions was adjusted using sodium hydroxide (NaOH) at a concentration of 0.1 M and hydrochloric acid (HCl) at a concentration of 0.05 M. The zeta potential was related to the electrophoretic mobility via Henry’s equation. The Hückel limit (f(κa) = 1.0) was applied for the calculations.

The measurements were performed for different BSA/5FU molar ratios: 1:10, 1:20, 1:40, and 1:60. The BSA concentration was constant at 0.25 mg/mL (3.8 μM). All complexes were prepared in distilled water, and the pH of all samples was adjusted to pH = 8.4. The BSA/5FU mixtures were stirred in the dark for 1 h. To remove uncomplexed drug molecules, the dialysis of complexes was performed using SnakeSkin™ dialysis tubes (MWCO 3.5 kDa, Thermo Fisher Scientific, Waltham, MA, USA). This process was carried out in distilled water at pH 8.4 for 24 h at room temperature.