Unveiling the interplay between homogeneous and heterogeneous catalytic mechanisms in copper-iron nanoparticles working under chemically relevant tumour conditions

Abstract

The present work sheds light on a generally overlooked issue in the emerging field of bio-orthogonal catalysis within tumour microenvironments (TMEs): the interplay between homogeneous and heterogeneous catalytic processes. In most cases, previous works dealing with nanoparticle-based catalysis in the TME focus on the effects obtained (e.g. tumour cell death) and attribute the results to heterogeneous processes alone. The specific mechanisms are rarely substantiated and, furthermore, the possibility of a significant contribution of homogeneous processes by leached species - and the complexes that they may form with biomolecules - is neither contemplated nor pursued. Herein, we have designed a bimetallic catalyst nanoparticle containing Cu and Fe species and we have been able to describe the whole picture in a more complex scenario where both homogeneous and heterogeneous processes are coupled and fostered under TME relevant chemical conditions. We investigate the preferential leaching of Cu ions in the presence of a TME overexpressed biomolecule such as glutathione (GSH). We demonstrate that these homogeneous processes initiated by the released by Cu-GSH interactions are in fact responsible for the greater part of the cell death effects found (GSH, a scavenger of reactive oxygen species, is depleted and highly active superoxide anions are generated in the same catalytic cycle). The remaining solid CuFe nanoparticle becomes an active catalyst to supply oxygen from oxygen reduced species, such as superoxide anions (by-product from GSH oxidation) and hydrogen peroxide, another species that is enriched in the TME. This activity is essential to sustain the homogeneous catalytic cycle in the oxygen-deprived tumour microenvironment. The combined heterogeneous-homogeneous mechanisms revealed themselves as highly efficient in selectively killing cancer cells, due to their higher GSH levels compared to healthy cell lines.

Fig. 1. Simplified overview of the homogeneous–heterogeneous processes fostered by the CuFe nanocatalyst in the presence of GSH. After a GSH-triggered Cu release from the nanocatalyst, Cu²⁺ catalyzes the homogeneous oxidation of GSH into GSSG. Simultaneously, Fe(iii) species present on the nanoparticle surface catalyze the conversion of H₂O₂ and ˙O₂⁻ species, considered as by-products from GSH oxidation into O₂ necessary to sustain the GSH depletion homogeneous cycle.

Fig. 2. GSH effect on the evolution of copper and iron cations lixiviated at different pH media: (a) pH = 7.4; (b) pH = 5.80; (c) different GSH ionic species as a function of different pH values; vertical lines represent the pH of selected experimental conditions for a better identification of expected GSH species; GSH concentration was set to 5 mM. Speciation diagram was generated using pK_a values obtained from.

Fig. 3. Homogeneous catalysis of ionic Cu and GSH with O₂. (a) Proposed homogeneous catalytic cycle for the Cu-assisted GSH oxidation. The reoxidation of Cu(SG)₂ to Cu(GSSG) involves a reaction between the Cu(GS–SG)˙⁻ complex and O₂, yielding superoxide radical species (˙O₂⁻) as reaction by-product. The shaded areas correspond to the structure of GS and GS(H)SG, which are abbreviated for a better understanding; (b) ¹H NMR spectra of GSH, GSSG, GSH + CuCl₂ and GSH + CuFe at different reaction times (2, 3 and 24 h) with the corresponding proton assignments. ¹H NRM signal at 3.22 ppm implies a chemical modification near –SH group of the native GSH molecule, either [Cu(SG)₂]⁺ or GSSG formation. ¹H NMR reaction spectra at 24 h is clearly altered due to paramagnetism induced by free Cu²⁺ in solution, since the reaction is complete and no GSH is available to coordinate Cu²⁺; (c) HRMS-ESI from control experiments with CuCl₂ + GSH binary mixture in anaerobic conditions to quench the catalytic reaction. Two peaks at m/z = 613.16 and 635.14 corresponding to [GSSG − H]⁺ and [GSSG + Na]⁺ confirmed the generation of GSH oxidation product. The catalytic intermediate Cu(SG)₂ is detected at m/z = 674 and 675, respectively; (d) evolution of GSH concentration in the presence of the CuFe nanocatalyst at pH 7.40 or 5.80 (adjusted with HCO₃⁻), 37 °C, [GSH]₀ = 5 mM; [CuFe] = 0.1 mg mL⁻¹; (e) influence of GSH on the generation of anion superoxide species ˙O₂⁻ as side-product of the Cu-catalyzed GSH oxidation. The absorbance of DPBF at a wavelength of 411 nm was used as indirect probe; reaction conditions: pH = 7.40 (adjusted with HCO₃⁻), [GSH]₀ = 5 mM, [DPBF]₀ = 0.1 mM, [CuFe] = 0.1 mg mL⁻¹.

Fig. 4. Heterogeneous catalysis on the Fe-enriched nanoparticle surface: (a) and (b) STEM-EDX images before/after catalyst interaction with GSH. Prior to the GSH-triggered lixiviation of Cu, Fe and Cu co-localize within the crystalline network of the nanoparticle oxide. However, after GSH exposition, Cu starts to be released and its presence in the nanoparticle is strongly reduced; (c) X-ray photoemission spectra corresponding to the Cu 2p_3/2 and Fe 2p regions before and after reaction of the CuFe nanocatalyst with GSH; as a consequence of leaching process, the intensity of Cu signal is close to noise; (d) TEM images of the CuFe nanocatalyst after 1 hour incubation with different GSH concentrations relevant at the intracellular and extracellular levels. Size analysis of individual nanoparticles reveals a certain reduction of size in the presence of larger GSH concentrations (5 mM); (e) scheme showing the transformation of the by-products generated via aerobic GSH oxidation into O₂ in the presence of the solid Fe-enriched catalyst that enables the regeneration of O₂ as electron acceptor to sustain the GSH oxidation cycle; (f) O₂ generation capabilities of the Fe-enriched nanoparticles and of the supernatant containing leached Cu cations after the addition of H₂O₂; experimental conditions: pH = 7.40; [H₂O₂]₀ = 1 mM, [CuFe] = 0.1 mg mL⁻¹; addition of H₂O₂ is highlighted.

Fig. 5. Cancer cell lines with overexpressed GSH levels are intensely affected by CuFe-triggered catalysis. (a) Left: scheme of catalyst evolution inside the cell, highlighting the role played by GSH; Right: intracellular GSH concentration levels detected for U251-MG, HeLa and hpMSC cell lines before and after 24 h of incubation with 25.0 μg mL⁻¹ of CuFe nanocatalyst. Statistical analysis revealed significant differences for cancer cells with higher GSH levels (i.e. U251-MG and HeLa) in comparison to hpMSC, which likely enhance GSH-related pathways, affecting cell viability; (b) cell viability study of CuFe nanocatalyst with different cancer (SKOV-3, U251-MG, HeLa and U87) and non-tumoral (hpMSC and fibroblasts) cell lines. CC50 values stand for the cytotoxic concentration to kill 50% of cell populations after 48 h of incubation; [CuFe] concentration for each experiment was (1) 12.5 μg mL⁻¹; (2) 25.0 μg mL⁻¹; (3) 50.0 μg mL⁻¹; (4) 100 μg mL⁻¹ and (5) 200 μg mL⁻¹; (c) confocal microscopy images corresponding to the U251-MG, HeLa and hpMSC cell lines before and after the incubation with CuFe NPs for 24 h using 25.0 μg mL⁻¹ of nanocatalyst; (actin fibres are displayed in red after staining with phalloidin-Alexa 488, nuclei are shown in blue and stained with DAPI and CuFe appear in green colour, pointed by yellow arrows and seen by reflection).

Fig. 6. Complete homogeneous-heterogeneous interplay for the CuFe nanoparticles in the presence of GSH and O₂: (i) in a first step, GSH triggers Cu-release from the spinel nanostructure (ii) excess GSH is able to form an organometallic complex with Cu through thiol (–SH) groups to stabilize Cu^I (iii) molecular O₂ accepts electrons from Cu(SG)₂ complex to yield O₂ reduced species (H₂O₂ or ˙O₂⁻, depending on the number of electrons transferred) and Cu(GSSG); (iv) in a heterogeneously coupled process, as-generated H₂O₂ and ˙O₂⁻ donates electrons to the remaining Fe-enriched surface of the solid heterogeneous nanocatalyst in a process that generates oxygen needed for step (iii). Moreover, intratumoural H₂O₂ decomposes on the Fe-enriched catalyst, contributing additional oxygen generation.