These data further confirm the presence of ionic interactions between amino group of basic amino acids in BSA and sulphate group of DS. Dissociation of HIP complex in presence of counter ions has also been reported by other investigators [13, 15]. Figure 2 Comparative dissociation of BSA from HIP complex in the presence of DI water and 10mM Na2HPO4 solution. FTIR study was performed to understand

the nature of interactions between amino group of basic amino acids in BSA and sulphate group of DS. FTIR analysis was performed by other investigators to Inhibitors,research,lifescience,medical characterize ionic interactions between oppositely charged functional groups [12, 23, 24]. Due to overlapping shift in a FTIR spectrum, we did not follow peak shift associated with the protein. Instead, the interaction of sulphate group of DS was studied in the IR region. Following are the characteristic peaks of sulphate group of DS in the IR region: (a) 802cm−1: S-O-S vibration, (b) 1017cm−1: symmetric SOO−stretching vibration, Inhibitors,research,lifescience,medical and (c) 1225cm−1: asymmetric SOO−stretching vibration. Appearance of these peaks Inhibitors,research,lifescience,medical in the IR spectra is close to previously published results [23–25]. Due to ionic interaction between amino and sulphate groups in HIP complex, the peak intensity of the sulphate group in the IR region may be attenuated

significantly. Results of this study are shown in Figure 3. These results clearly indicate a significant reduction in the peak intensities of sulphate group in the IR region which again confirmed the presence of ionic interactions between amino and sulphate groups in the HIP complex. Figure 3 FTIR spectra of Inhibitors,research,lifescience,medical (a) BSA, (b), DS and (c) HIP complex. We prepared nanoparticles of the complex using S/O/W emulsion method. This method of preparation offers significant advantages over conventional methods of nanoparticles preparation such as single and double emulsion method. In the

conventional methods of preparation, protein is initially dissolved Inhibitors,research,lifescience,medical in an aqueous phase and later emulsified in the presence of an organic phase using sonication. Most protein denaturation BMS-345541 clinical trial occurs during this stage of nanoparticle because preparation due to water-organic phase interface. Excessive stress during sonication process and generation of free radicals can cause protein unfolding and denaturation. In S/O/W emulsion method, protein-polysaccharide powder was employed in the preparation of nanoparticles instead of protein in solution form. Further, in the powder form, kinetic mobility of the protein is restricted compared to solution form [20, 21]. Moreover, complexation with DS would not only restrict conformational flexibility of BSA but would also impart additional steric shielding to the protein molecule. We optimized the total volume of organic solvent needed and the sonication time to prepare nanoparticles.