Adsorption of hemoglobin onto gold nanoparticles: role of ligand coating on assembly formation and protein structure

Understanding the interaction of hemoglobin (Hb) with various nanomaterials and nanoparticles (NPs) is of considerable interest for the design of enhanced oxygen carriers for therapeutic applications. In this report, we undertake an iterative study of the role of the capping ligand in determining the nature of Hb binding to the surface of spherical gold nanoparticles (AuNPs, 5 nm diameter). We show that AuNPs capped with citrate and thioctic acid TA-SO₃ ligands show robust assembly of Hb to the AuNP surface while NPs capped with TA-CL4 ligands (short, zwitterionic ligands) exhibit minimal assembly of Hb to the NP surface. Citrate on the AuNP facilitates the efficient assembly of Hb to the NP surface through the formation of an Au–S bond between Au and free thiols on cysteine residues. Conversely, TA-SO₃-capped AuNPs mediate the electrostatic assembly of Hb to the ligand coating. Hb assembly on the citrate and TA-SO₃ AuNPs significantly changes the AuNP physical properties (e.g., hydrodynamic diameter, surface charge, and colloidal stability). Hb assembly on the citrate-AuNPs alters its spectral properties, while Hb assembly to TA-SO₃-AuNPs shows minimal changes in AuNP spectral properties. More importantly, Hb undergoes different degrees of spectral and interstitial structural alterations, including changes to the heme absorption peak and modification in the secondary structure, upon being assembled with the AuNP. Circular dichroism (CD) spectroscopy data confirms a significant (50%) loss of Hb α-helix structure when assembled on the citrate-AuNP compared with TA-SO₃-AuNP. Our results demonstrate the important consideration that must be given to the ligand coating when interfacing Hb (and other proteins) with NP materials for bioconjugate formation.