Assessment of gold nanoparticles on human peripheral blood cells by metabolic profiling with 1H-NMR spectroscopy, a novel translational approach on a patient-specific basis

Abstract

Human peripheral blood cells are relevant ex vivo models for characterizing diseases and evaluating the pharmacological effects of therapeutic interventions, as they provide a close reflection of an individual pathophysiological state. In this work, a new approach to evaluate the impact of nanoparticles on the three main fractions of human peripheral blood cells by nuclear magnetic resonance spectroscopy is shown. Thus, a comprehensive protocol has been set-up including the separation of blood cells, their in vitro treatment with nanoparticles and the extraction and characterization of metabolites by nuclear magnetic resonance. This method was applied to assess the effect of gold nanoparticles, either coated with chitosan or supported on ceria, on peripheral blood cells from healthy individuals. A clear antioxidant effect was observed for chitosan-coated gold nanoparticles by a significant increase in reduced glutathione, that was much less pronounced for gold-cerium nanoparticles. In addition, the analysis revealed significant alterations of several other pathways, which were stronger for gold-cerium nanoparticles. These results are in accordance with the toxicological data previously reported for these materials, confirming the value of the current methodology.

Fig 1. Strategy for peripheral blood cell isolation (PBMCs, PMNs and erythrocytes), nanoparticle treatment, polar and no-polar metabolite extraction, and ¹H-NMR analysis.

PBMCs, PMNs and erythrocytes samples were isolated from peripheral blood of healthy human individuals. Samples of each cell type were split an aliquot (20 million cells) for characterization (a), and another aliquot (40 million cells) for nanoparticle treatments (b). Finally, polar and nonpolar were extracted and the ¹H-NMR metabolic profiles determined (c).

Fig 2. ¹H-NMR spectra of erythrocytes, PMNs and PBMCs.

Polar (a) and non polar (b) ¹H-NMR metabolomic profiles of extracts of the main types of peripheral blood cells. Metabolite assignments are indicated with the following numbers: 1) 2-hydroxybutyrate, 2) leucine, 3) valine, 4) ethanol, 5) lactate, 6) 2-aminoisobutyrate, 7) alanine, 8) lysine, 9) acetate, 10) glutamate, 11) reduced glutathione (GSH), 12) oxidized glutathione (GSSG), 13) pyroglutamate, 14) pyruvate, 15) succinate, 16) glutamine, 17) creatine, 18) phosphocreatine, 19) malonate, 20) spermidine/spermine, 21) phosphocholine (PC), 22) glycerophosphocholine (GPC), 23) carnitine, 24) betaine, 25) taurine, 26) methanol, 27) proline, 28) glycine, 29) glycerol, 30) ascorbate, 31) guanidino/guanido acetate, 32) 6-phosphogluconate 33) glycolate, 34) phosphoethanolamine, 35) ATP, 37) AMP, 38) NAD+, 39) NADP+, 40) trehalose, 41) phosphoenolpyruvate, 42) UDP-glucose, 43) UDP-NAG, 43) NADH, 44) uracil/tryptophane, 45) GDP, 46) GTP, 47) NADPH, 48) CTP, 49) fumarate, 50) tyrosine, 51) histidine/histamine, 52) tryptophane, 53) phenylalanine, 54) guanosine, 55) xanthine, 56) guanine, 57) hypoxanthine, 58) CTP, 59) CDP, 60) ADP, 61) formate, 62) methionine, 63) aspartate, 64) malate, 65) glucose, 66) isoleucine, 67) acetoacetate, 68) methylacetoacetate, 69) sarcosine, 71) thymidine, 72) hydroquinone, 73) pyridoxamine, 74) 4-pyridoxate, 75) nicotinamide, 76) N-Methyl-a-aminoisobutyric acid, 80) cholesterol, 81) lipid CH₃-, 82) lipid -CH₂-, 83) fatty ester -CH₂CH₂COO-, 84) polyinsaturated fatty acids (PUFA), 85) monoinsaturated fatty acids (MUFA), 86) phosphatidylethanolamine, 87) phosphatidycholine, 88) acylglycerophosphoserine, 89) phospholipids, 90) TAG, 91) spingosine, 92) fatty ester—CH₂OCO-.

Fig 3. ¹H NMR spectra of the aqueous extracts of RBCs, PMNs and PBMCs after treatment with nanoparticles.

Changes observed in the metabolomic profile of the main peripheral blood cells after 3h of treatment with vehicle, AuChi and AuCeO₂ nanoparticles.