Insights into the Thermodynamics of Polymer Nanodot–Human Serum Albumin Association: A Spectroscopic and Calorimetric Approach

Abstract

With the advent of newer luminescent nanoparticles for bioimaging applications, their complex interactions with individual biomolecules need to be understood in great detail, before their direct application into cellular environments. Here, we have presented a systematic and detailed study on the interaction between luminescent polymer nanodots (PNDs) and human serum albumin (HSA) in its free and ligand-bound state with the help of spectrophotometric and calorimetric techniques. At physiological pH (pH = 7.4), PNDs quench the intrinsic fluorescence of HSA as a consequence of ground-state complex formation. The binding stoichiometry and various thermodynamic parameters have been evaluated by using isothermal titration calorimetry and the van't Hoff equation. It has been found that the association of PNDs with HSA is spontaneous (ΔG = -32.48 ± 1.24 kJ mol) and is driven by a favorable negative standard enthalpy change (ΔH = -52.86 ± 2.12 kJ mol) and an unfavorable negative standard entropy change (ΔS = -68.38 ± 2.96 J mol K). These results have been explained by considering hydrogen bonding interactions between amino and hydroxyl groups (-NH and -OH) of PNDs and carboxylate groups (-COO) of glutamate (Glu) and aspartate (Asp) residues of HSA. The binding constant of PNDs with HSA is estimated to be 4.90 ± 0.19 × 10 M. Moreover, it has been observed that warfarin-bound HSA (war-HSA) shows a significantly lower binding affinity (K = 1.15 ± 0.19 × 10 M) toward PNDs, whereas ibuprofen-bound HSA (ibu-HSA) shows a slightly lower affinity (K = 3.47 ± 0.13 × 10 M) compared with the free HSA. In addition, our results revealed that PNDs displace warfarin from site I (subdomain IIA) of HSA because of the partial unfolding of war-HSA. We hope that the present study will be helpful to understand the fundamental interactions of these biocompatible PNDs with various biological macromolecules.

Investigating the Interaction of Nanodiamonds with Human Serum Albumin and Induced Cytotoxicity

“…At physiological pH 7.0, the carboxyl groups were deprotonated and induced the negatively charged surfaces of NDs [23]. Furthermore, the isoelectric point (pI) of HSA is ∼4.7 and thus HSA shows negatively charged surface at pH 7.0 [14]. erefore, the electrostatic interactions and hydrophobic interaction between HSA and carboxylated NDs can be eliminated, which coincides with the results of thermodynamic analysis.…”

“…e mean diameter decreased to 73 nm in the presence of HSA (Figure 1(d)). e changing trends of DLS coincide with the results in TEM, which suggests that the interaction between HSA and NDs may be ascribed to the static ground-state association [14]. Figure 2(a) shows the absorption spectra of NDs, HSA, and HSA-NDs.…”

“…To investigate the conformation of HSA, circular dichroism (CD) was performed to monitor the secondary structure of HSA in the presence of NDs. As shown in Figure 2(b)) native HSA exhibits two negative absorption bands at 208 nm and 222 nm, which are assigned to the parallel exactions of the π − π * transition of peptide and the n − π * transition of the carbonyl group, respectively [14,26]. e α-helix content of HSA is estimated to be 51.7%.…”

“…A fluorescence quenching technique has been effectively and extensively applied to investigate the biding mechanism and conformation of protein interacting with nanomaterials [31]. As shown in Figure 3(a), HSA shows an intrinsic fluorescence at 336 nm mainly due to the presence of Trp-214 in subdomain IIA [14]. e intrinsic fluorescence of HSA is progressively quenched by the presence of NDs with increasing concentration, which reveals the interacting characteristics of HSA on NDs' surface.…”

“…Human serum albumin (HSA) is the most abundant protein in the circulatory system and possesses specific transportation and deposition of a variety of endogenous ligands, such as fatty acids, and exogenous ligands, such as metal ions, nanoparticles, and a variety of therapeutics drugs [12,13]. Interacting with nanoparticles, HSA forms the first layer of the long-lived hard corona on the surface of nanoparticles, which increases their circulatory half-lives in the blood and improves distribution and biocompatibility throughout the body [14]. Several previous reports have investigated the interactions of various nanomaterials with HSA and the induced structure change and stability of proteins [15][16][17].…”