HIP complexation based approach can be explored to deliver peptide and protein-based therapeutics. It can overcome various stability related issues, enhance drug loading in nanocarriers and improve drug permeation across biological membrane [10–14, 22]. So far, HIP complex based approach has been only studied with small peptide and protein-based therapeutics. Hence, BSA was selected as a model mTOR inhibitor protein in the present study because of its higher molecular weight (66.3kDa) and well-known secondary and tertiary structure. Isoelectric

point (pI) of BSA Inhibitors,research,lifescience,medical is ≈4.5, and the protein consists of various basic amino acids (60 lysine and 26 arginine residues). Hence, we have slightly altered the pH of BSA solution and prepared

stock solution of BSA at pH 4.4 in citrate buffer. Being hydrophilic in nature, these amino acids are mostly found on the protein surface. Amino groups of these basic amino acids Inhibitors,research,lifescience,medical are protonated based on the pH of surrounding medium. At this pH, HIP complex was formed immediately upon mixing of aqueous solutions of BSA and DS. This data confirms the importance Inhibitors,research,lifescience,medical of pH of the protein solution prior to HIP complexation. In general, it is crucial to understand the effect of pH on stability of protein molecule. One should also consider the possibility of other stability related issues which may arise by changing the pH of protein solution prior HIP complexation. The effect of molar ratios of DS/BSA on HIP complex formation has been studied. We calculated the molar ratios based on the total number of lysine amino acids present on the surface of BSA (60 lysine amino acid). HIP complexes were prepared using the following molar Inhibitors,research,lifescience,medical ratios

of DS/BSA (0.29, 0.58, 0.87, and 1.15). Theoretically, these molar ratios represent the amounts of DS added which was sufficient to complex with 15, 30, 45, Inhibitors,research,lifescience,medical and 60 basic amino acids of BSA. Figure 1 shows the complexation of BSA with DS at different molar ratios.An excellent correlation is observed between increments in the molar ADAMTS5 ratio of DS/BSA with the amount of BSA complexed with DS (Figure 1).In fact at a molar ratio of 1.15, more than 90% of BSA molecules were ionically complexed with DS. This data clearly indicates the involvement of basic amino acids in the formation of HIP complex. Figure 1 Effect of molar ratio of DS:BSA on HIP complex formation. We also hypothesized ionic interactions as a driving force for complexation of BSA with DS. In order to confirm our hypothesis, we performed dissociation studies of the HIP complex in presence of oppositely charged ions (HPO4−2). Results of this experiment are shown in Figure 2. When HIP complex was incubated in DI water, no dissociation of BSA from HIP complex was observed. This could be due to low ionic strength of DI water.