25 mg/mL de pepsin at 37°C overnight following with a centrifugation and dialysis against TBS (145 mM NaCl and 10 mM Tris, pH 7.5) for 18 h. The Staph A-Sepharose column (Pharmacia, Kalamazoo, MI, USA) was used to remove the undigested mAbs at pH 8.0 resulting in the rituximab F(ab)2. The obtained

F(ab)2 was further purified by the Sephadex G-150 column (Pharmacia, MI, USA) which was pre-equilibrated by the buffer 1 (0.1 M NaCl, 0.1 M borate, 0.05 M citrate and 2 mM EDTA, pH 5.5). Such F(ab)2 solution was concentrated to 10 mg/mL and further digested by the enzyme papain. The Fab fragment solution was https://www.selleckchem.com/products/ly2874455.html purified by the same procedure as mentioned above resulting in the single Fab fragment stocking solution storing at 4°C. To activate the Fab fragments of rituximab for reactivity toward the maleimide, the above stocking solutions were incubated with 2-iminothiolane (2-IT, Sigma-Aldrich, St. Louis, MO, USA) with a mass ratio of 1:0.15 (Fab/2-IT) at room temperature for 2 h under a gentle shake. Unreacted 2-IT was removed by dialysis. The bovine serum albumin Selleckchem FK506 (BSA) ~ SH was produced in the same way. The resulting reactive Fab ~ SH and BSA ~ SH were stored at 4°C for future usage [28]. Fabrication of rituximab Fab-conjugated liposome The Fab fragment-conjugated liposome was prepared by

coupling the reactive Fab ~ SH onto the liposomal surface via the reaction between the ~ SH and Mal-group at 4°C and N2 environment overnight; the un-conjugated Fabs were removed

by dialysis. The BSA-conjugated liposome was fabricated in the same way. For UV irradiation, pure liposome solutions were exposed to 20 irradiation cycles at 4°C, with a 254-nm UV light dose of 360 mJ/cm2 per cycle using a Stratalinker-UV 1800 [26]. The concentration of Fab fragments in the liposome solution was quantified by measuring the A260/A280 using Nano VueTM (GE Healthcare, Morin Hydrate Wilmington, MA, USA). Characterization of Fab fragment-conjugated liposome The hydrodynamic diameter and size distribution were determined by ZetaSizer (Nano-ZS, Malvern Instruments, Worcestershire, UK) equipped with a HeeNe laser (633 nm) at the scattering angle 173°. To prepare stained specimens for TEM (H-7000 click here Electron Microscope, Hitachi, Tokyo, Japan) experiments, about 5 μL liposome solution was dropped on 200-mesh Formvar-free carbon-coated copper grids (Ted Pella Type-A; nominal carbon thickness 2 to 3 nm). After the water evaporating by exposing to air at room temperature, the sample was inversely covered on a small drop of hydrodated phosphotungstate (PTA) solution with a mass fraction of 2%. The conventional TEM images were obtained at 100 kV. Weight-average molecular weight analysis by SLS The static light scattering (SLS) measurements were carried out varying the scattering angle (θ) from 40 to 140° with a 5° stepwise increase [29].