Addressing the functional biomaterials as next-generation therapeutics, chitosan and alginic acid were copolymerized in the form of chemically crosslinked interpenetrating networks (IPNs). The native hydrogel was functionalized via carbodiimide (EDC), catalyzed coupling of soft ligand (1,2-Ethylenediamine) and hard ligand (4-aminophenol) to replace -OH groups in alginic acid units for extended hydrogel- interfaces with the aqueous and sparingly soluble drug solutions. The chemical structure, Lower solution critical temperature (LCST ≈ 37.88 °C), particle size (Zh,app ≈ 150-200 nm), grain size (160-360 nm), surface roughness (85-250 nm), conductivity (37-74 mv) and zeta potential (16-32 mv) of native and functionalized hydrogel were investigated by using FT-IR, solid state-13C-NMR, TGA, DSC, FESEM, AFM and dynamic light scattering (DLS) measurements. The effective swelling, drug loading (47-78%) and drug release (53-86%) profiles were adjusted based on selective functionalization of hydrophobic IPNs due to electrostatic complexation and extended interactions of hydrophilic ligands with the aqueous and drug solutions. Drug release from the hydrogel matrices with diffusion coefficient n ≈ 0.7 was established by Non- Fickian diffusion mechanism. In vitro degradation trials of the hydrogel with a 20% loss of wet mass in simulated gastric fluid (SGF) and 38% loss of wet mass in simulated intestinal fluid (SIF), were investigated for 400 h through bulk erosion. Consequently, a slower rate of drug loading and release was observed for native hydrogel, due to stronger H-bonding, interlocking and entanglement within the IPNs, which was finely tuned and extended by the induced hydrophilic and functional ligands. In the light of induced hydrophilicity, such functional hydrogel could be highly attractive for extended release of sparingly soluble drugs.
The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl β-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species.