This was achieved by stirring one volume of 2% (w/v) alginate solution for 20 min with one-half Selleck Omipalisib volume of 0·08% (w/v) 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC-HCl) and 3% (w/v) sulfo-NHS solution. The resulting mixture was incubated for 17 h at room temperature with one volume of

alfa-t-butyloxycarbonylamino-omega-amino poly (ethylene glycol) PEG (MW 5000 Da). After dialysis using a tubular membrane (100 kDa MWCO, Spectra/Por® Biotech Cellulose Ester; Spectrum Ls Europe B.V., Breda, The Netherlands) against 1000 volumes of demineralized water, the product was freeze-dried, weighed and placed in flat bottom beaker to be completely covered for 40 min at room temperature by trifluoroacetic acid (TFA; Fluka Sigma-Aldrich Ltd). Thereafter, the TFA was removed under a nitrogen

flow, and the product finally freeze-dried overnight. The alginate-PEG5k-NH2 (modification rate 1 : 50 units) obtained was dissolved at 2 mg/mL in carbonate buffer 0·1 m pH 9·0 (freshly prepared). One volume of 0·1% (w/v) α-d-mannopyranosyl-phenyl isothiocyanate (Fluka Sigma-Aldrich Ltd) in DMSO was then added drop-wise with constant agitation to 50 volumes of alginate (theoretical modification rate was 1 : 50 units). After approximately 30- min agitation, the solution was stored overnight at 4°C. The suspension was then dialysed with a 100 kDa MWCO membrane (Spectrum Ls Europe B.V.) against 300 volumes demineralized H2O. The filtrate was changed four times every 2 h, and the product freeze-dried buy SP600125 for storing at −20°C. Mannose-alginate decorated nanogels were prepared as described in Nanogel surface decoration with alginate, using this alginate-mannose instead of alginate. The final concentration of recNcPDI in the nanogel suspension after concentration was 50 μg PDI/mL dispersion. Recombinant NcPDI and recNcPDI-nanogel preparations were subjected

to ultracentrifugation (150 000 × g, 25 min, 4°C) using a TST55.5 rotor and a Centrikom T-2070 ultracentrifuge. The association of recNcPDI antigen with the nanogels was evaluated by analysing supernatant and pellet fractions by 12·5% (w/v) sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), carried out under reducing conditions following boiling of the Y-27632 2HCl samples in sample buffer (40). Protein bands were visualized by silver staining and Western blotting as previously described (40). For immunoblotting, rat anti-recNcPDI (18) diluted 1 : 1000 in PBS containing 0·3% (w/v) BSA was used. The secondary antibody was an anti-rat IgG alkaline phosphatase conjugate (Promega, Madison, USA), which was applied according to the instructions provided by the manufacturer. One hundred and thirty female Balb/c mice (6 weeks of age) were purchased from Charles River Laboratories (Sulzheim, Germany) and were housed under conventional day/night conditions according to the standards set up by the animal welfare legislation of the Swiss Veterinary Office.