Development of novel formulations for mucosal delivery of protein ...

3.2 Materials and methods
3.2.1 Materials
Table 3.1: Materials
Material
Description/Batch Supplier
L-cysteine hydrochloride C1276-10G Sigma, (Gillingham, UK)
5,5-Dithiobis (2-nitrobenzoic D8130-5G Sigma-Aldrich, (Gillingham, UK)
acid) (Ellman’s reagent)
Pullulan 200,000 standard 01615-25MG Sigma-Aldrich, (Gillingham, UK)
Sodium acetate
W302406-1KG-K Sigma-Aldrich, (Gillingham, UK)
Bradford’s reagent B6916-500ML Sigma-Aldrich, (Gillingham, UK)
BSA powder A7906-100 G Sigma-Aldrich, (Gillingham, UK)
Acetic acid A /0360/PB17 Fisher Scientific (Leicester, UK)
PBS tablets pH 7.4 Sigma-Aldrich, (Gillingham, UK)
Human INS 12643-50MG Sigma-Aldrich, (Gillingham, UK)
Acetonitrile
34967-2.5L Sigma-Aldrich, (Gillingham, UK)
(Fluka)
Copper(II) chloride 451665-5G Sigma-Aldrich, (Gillingham, UK)
Trifluoroacetic acid (TFA) 302031-100ML Sigma-Aldrich, (Gillingham, UK)
Potassium dihydrogen
phosphate
P /4760/53 Fisher Scientific(Leicester UK)
3.2.2 Structural elucidation of chitosan and TG-chitosan- 1 H-NMR
Proton nuclear magnetic resonance ( 1 H-NMR) spectra were acquired using a Jeol
ECA, 500 MHz FT NMR spectrometer, incorporating a NM-50TH5AT/FG2 probe (Tokyo,
Japan). Chitosan powder and TG-chitosan were dissolved in 2 % deuterium chloride (DCl)
solution in D 2 O and placed in a 5 mm borosilicate glass NMR tube and spun at 15 Hz. 1 H
spectra were acquired using a single pulse experiment and the 1 H –NMR parameters are
summarised in Table 3.2. The degree of acetylation (DA) was determined from equation 3.1
DA = (I CH3 /3)/(I H2-H6 /6) x 100 % equation 3.1
Where I CH3 is the integral intensity of N-acetyl protons and I H2-H6 is the sum of the integral
intensities of H-2. -3, -4, -5 and H-6 of the acetylated rings (Sogia et al., 2008).
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