PEGylated gold nanoparticles regulate metabolic flux in erythrocytes by inducing hemoglobin deoxygenation

Nanomedicines based on polyethylene glycol-functionalized gold nanoparticles (Au@PEG NPs) are usually administered by intravenous injection to improve bioavailability. It is widely accepted that surface modification with PEG can prevent direct interactions between AuNPs and proteins. Therefore, the interaction of Au@PEG NPs with plasma proteins and blood cells has not received enough attention. Our previous study demonstrated that Au@PEG NPs affect the oxygen-carrying capacity and deformability of erythrocytes through oxidative stress; however, the molecular mechanism of oxidative damage induced by Au@PEG NPs remains unclear. Due to the absence of cell organelles such as the nucleus and mitochondria in mature erythrocytes, we hypothesise that Au@PEG NPs primarily generate oxidative stress by interfere with metabolic flux in erythrocytes. We have employed dynamic light scattering (DLS), isothermal titration calorimetry (ITC) and surface plasmon resonance imaging (SPRi) to investigate the interaction between proteins and 30 nm Au@PEG NPs. ITC and SPRi data revealed that hemoglobin exhibits a higher affinity for 30 nm Au@PEG NPs compared to albumin (Ka: 1.46 × 10^-4 <i>vs</i> 1.08 × 10^-4 M^-1). Circular dichroism (CD) spectrum demonstrated a significant conformational shift in hemoglobin following incubation with 30 nm Au@PEG NPs, characterized by an increase in α-helix [(65.9 ± 0.6) % to (68.2 ± 0.6) %] and a decrease in β-sheet [(4.8 ± 0.3) % to (1.7 ± 0.2) %], which is consistent with its transition toward a deoxygenated state. By combining ICP-MS and four specific endocytosis inhibitors, we investigated the endocytic mechanism of Au@PEG NPs entering erythrocytes. The data revealed that the uptake efficiency of 30 nm Au@PEG NPs by erythrocytes was 68.2 ± 0.6 ng/10⁹ RBCs, with approximately 75% of the Au@PEG NPs were uptaked by erythrocytes through a mechanism involving caveolin- and clathrin-mediated endocytosis. Metabolomics and NADP⁺/NADPH analysis revealed that Au@PEG NPs induce hemoglobin deoxygenation, which in turn inhibits the pentose phosphate pathway and disrupts redox homeostasis, as reflected by the decrease of intracellular NADPH from 3.53 ± 0.50 nmol/10¹² RBCs to 2.67 ± 0.46 nmol/10¹² RBCs. However, tail vein injection of Au@PEG NPs did not impair the liver oxygen supply since mice can compensate for the hemoglobin deoxygenation effect of Au@PEG NPs by decreasing systolic blood pressure and increasing tissue perfusion. Our findings showed that Au@PEG NPs interfere with metabolic flux and generate oxidative stress in erythrocytes by changing the oxygenation state of hemoglobin, and these results suggest that future studies should pay more attention to hemocompatibility evaluations of nanomedicines before their clinical application.