Sodium triacetoxyborohydride

The following materials and reagents were used for synthesis: silica gel 60 (70–230 mesh) was purchased from Merck GmbH (Darmstadt, Germany), 3-fluoroaniline, (R)-glycidyl butyrate, n-buthyllithium (1.6 M in hexane), sodium azide, triethylamine, methanesulphonyl chloride, trifluoroacetic acid silver salt, thioacetic acid, iodine, tetrakis(triphenylphosphine)palladium(0), 4N hydrogen chloride in 1,4-dioxane and trifluoroacetic acid, propargylamine, 4-formylphenylboronic acid, sodium triacetoxyborohydride, and di-tert-butyl dicarbonate were purchased from Sigma-Aldrich (Steinheim, Germany), 4-methoxybenzyl chloride, benzyl chlorformate were purchased from TCI (Tokyo, Japan), sodium bicarbonate, magnesium sulphate, sodium sulphate, potassium carbonate, sodium chloride, ammonium chloride, hexane, ethyl acetate, methanol, acetone, tetrahydrofuran, hydrochloric acid, ethanol, toluene, dichloromethane, and chloroform were purchased from POCH (Gliwice, Poland) and all were of analytical grade. N,N-dimethylformamide, and acetonitrile were purchased from Rathburn Chemicals (Walkerburn, Great Britain) and were of HPLC grade.
The reference standard of RAD was purchased from ApexBio Technology LLC (Houston, TX, USA).

chitosan-based NPs encapsulating KAg (CS NPs-KAg) and mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccines were prepared using an ionic gelation method as previously described (24 (link)). For mannose-conjugated chitosan (mCS) preparation, 40 mg each of mannose (Sigma, MO) and sodium triacetoxyborohydride (Sigma, MO) mixture in 0.2 M borate buffer was slowly added into 200 mg chitosan [1% (w/v)] suspension (25 (link)) under magnetic stirring for 72 h at 56°C (34 (link)). The mCS was dialyzed against milli-Q-water for 48 h. Both chitosan and mannose modified chitosan were dissolved in 1% acetic acid solution. Twenty milligrams of mCS or chitosan were added into 20 ml milli-Q-water under magnetic stirring, pH was adjusted to 4.3 and mixed with 2 mg KAg (H1N2-OH10) in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer pH 7.4. Followed by tripolyphosphate [1% (w/v) (Sigma, MO)] 5 mg in 10 ml milli-Q-water was added dropwise and the mCS NPs-KAg or CS NPs-KAg vaccines were obtained after centrifugation at 10,976 × g for 30 min, washed, dispersed in milli-Q-water and used for vaccination. Both the vaccines had ~80% antigen encapsulation efficiency and characterized as described previously (24 (link), 25 (link)). Both the chitosan-based vaccine formulations were freshly prepared and used in animals.

Medium molecular weight chitosan (88.3% deacetylated; CAS number: 9012-76-4; Lot No.: 281219) was obtained from ChitOcean Inc. (St John’s, NL, Canada). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), non-essential amino acid (NEAA, product code 11140035), Hank’s balanced salt solution (HBSS, H8264), 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 0.25% (w/v) trypsin, ethylenediaminetetraacetic acid (EDTA) solution (0.02% in 0.5 mM DPBS), Dulbecco’s modified eagle medium (DMEM, product code D6546), penicillin-streptomycin (product code P4333), L-glutamine, D-(+)-mannose (product code M8574), sodium pentatripolyphosphate (TPP, product code 72061), sodium triacetoxyborohydride (product code 316393), coumarin-6 (C6, product code 442631), and Triton^®-X-100 were purchased from Sigma Aldrich (Arklow, Ireland).