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. 2023 May 23;13(23):15540-15553.
doi: 10.1039/d3ra00689a. eCollection 2023 May 22.

Hemoglobin-stabilized gold nanoclusters displaying oxygen transport ability, self-antioxidation, auto-fluorescence properties and long-term storage potential

Xingli Cun  1 , Michelle M T Jansman  1 , Xiaoli Liu  1 , Victor Boureau  2 , Peter W Thulstrup  3 , Leticia Hosta-Rigau  1
Affiliations

Hemoglobin-stabilized gold nanoclusters displaying oxygen transport ability, self-antioxidation, auto-fluorescence properties and long-term storage potential

Xingli Cun et al. RSC Adv. .

Abstract

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) holds a lot of potential to overcome important drawbacks of donor blood such as a short shelf life or the potential risk of infection. However, a crucial limitation of current HBOCs is the autoxidation of Hb into methemoglobin (metHb), which lacks oxygen-carrying capacity. Herein, we address this challenge by fabricating a Hb and gold nanoclusters (AuNCs) composite (Hb@AuNCs) which preserves the exceptional features of both systems. Specifically, the Hb@AuNCs retain the oxygen-transporting properties of Hb, while the AuNCs provide antioxidant functionality as shown by their ability to catalytically deplete harmful reactive oxygen species (ROS). Importantly, these ROS-scavenging properties translate into antioxidant protection by minimizing the autoxidation of Hb into non-functional metHb. Furthermore, the AuNCs render Hb@AuNCs with auto-fluorescence properties which could potentially allow them to be monitored once administered into the body. Last but not least, these three features (i.e., oxygen transport, antioxidant and fluorescence properties) are well maintained following storage as a freeze-dried product. Thus, overall, the as-prepared Hb@AuNCs hold the potential to be used as a multifunctional blood surrogate in the near future.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Scheme 1
Scheme 1. Schematic illustration of the as-prepared Hb-protected ultra-small Au nanoclusters and their multiple functions. Within the Hb@AuNCs, the Hb provides oxygen (O2) binding and releasing properties, while the AuNCs provide autofluorescence properties and antioxidant catalytic activity against harmful reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and against methemoglobin (metHb) conversion.
Fig. 1
Fig. 1. Characterization of Hb@AuNCs. (A) Photographic image of free Hb (left) and Hb@AuNCs (right). (B) UV-vis absorption (Abs) spectrum of Hb@AuNCs. (C) Fluorescence intensity (FI) excitation (i) and emission (ii) spectra of Hb@AuNCs. The FI emission spectrum has been obtained using an excitation wavelength (λex) of 494 nm. (D) FI emission spectra of Hb@AuNCs excited at different wavelengths.
Fig. 2
Fig. 2. Characterization of Hb@AuNCs. (A) Size, polydispersity index (PDI) and zeta (ζ)-potential of free Hb and Hb@AuNCs and the corresponding size distributions. (B) Scanning transmission electron microscopy analysis of Hb@AuNCs showing an annular dark-field image (i) and the corresponding energy dispersive X-ray elemental map of Fe (red) and Au (yellow) (ii).
Fig. 3
Fig. 3. Structural characterization of Hb@AuNCs. (A) FTIR and the corresponding second derivative spectra (B) of free Hb and Hb@AuNCs. (C) (i) Quantitative contribution of the different structural elements to the secondary structures of Hb and the Hb@AuNCs. (ii) Ratios of β-sheets/α-helix and intramolecular/intermolecular aggregates (intramolec./intermolec. aggreg.) present in Hb and Hb@AuNCs.
Fig. 4
Fig. 4. Structural characterization of Hb@AuNCs. (A) Circular dichroism spectra of Hb and Hb@AuNCs. (B) Contribution of the different structural elements to the secondary structure of Hb and the Hb@AuNCs.
Fig. 5
Fig. 5. Oxygen binding and releasing capacity of Hb@AuNCs. UV-vis spectra of free Hb (A) and Hb@AuNCs (B) after three subsequent cycles of purging with compressed air and nitrogen (N2) gas. The table shows the wavelengths of both free Hb and Hb@AuNCs after each cycle.
Fig. 6
Fig. 6. Fluorescent properties of Hb@AuNCs. Fluorescence excitation (A) and emission spectra (B) of Hb@AuNCs after each cycle of purging with compressed air or nitrogen (N2) gas. (C) Fluorescence intensity (FI) readings of Hb@AuNCs after each cycle of purging. Free Hb at the same concentration was set as controls. Data represent mean ± SD (n = 3).
Fig. 7
Fig. 7. Catalase-like activity of Hb@AuNCs evaluated by the Amplex Red assay. (A) Hydrogen peroxide (H2O2) in the presence of horseradish peroxidase (HRP) oxidizes the Amplex Red probe into the fluorescent resorufin product giving water (H2O) and molecular oxygen (O2) as by-products. (B) Normalized fluorescence intensity (nFI) readings due to the fluorescent resorufin product following incubation of BSA@AuNCs and Hb@AuNCs with 4.3 μM H2O2 for several time intervals at two concentrations: high concentration (HC): 0.25 mg mL−1 or 0.5 mg mL−1 for BSA@AuNCs and Hb@AuNCs, respectively, and low concentration (LC): 0.125 mg mL−1 or 0.25 mg mL−1 for BSA@AuNCs and Hb@AuNCs, respectively. (C) nFI readings due to the resorufin product for four subsequent cycles. Data represent mean ± SD (n = 3).
Fig. 8
Fig. 8. Antioxidant properties of Hb@AuNCs. Normalized Soret peak height over time of free Hb and Hb@AuNCs. The samples were incubated in a 0.09 mM hydrogen peroxide (H2O2) solution.
Fig. 9
Fig. 9. Storage stability of Hb@AuNCs. (A) UV-vis spectra of re-dispersed Hb@AuNCs after three subsequent cycles of purging with compressed air and nitrogen (N2) gas. The table shows the wavelengths of the main peaks after each cycle. (B) Fluorescence excitation (i) and emission (ii) spectra of freshly prepared and freeze-dried Hb@AuNCs. The emission spectra was obtained using an excitation wavelength (λex) of 494 nm. (iii) Fluorescence intensity (FI) readings of Hb@AuNCs after preparation and after being re-dispersed. Free Hb was set as a control. (C) Normalized Soret peak height over time of free Hb, Hb@AuNCs and re-dispersed Hb@AuNCs. The samples were incubated in a 0.09 mM hydrogen peroxide solution. The re-dispersed Hb@AuNCs had been stored as a powder for 14 days at 25 °C. Data represent mean ± SD (n = 3).
Fig. 10
Fig. 10. Characterization of MOF-NPs. (A) Size, polydispersity index (PDI) and zeta (ζ)-potential of bare MOF-NPs, Hb-loaded MOF-NPs (MOF(Hb)-NPs) and Hb@AuNCs-loaded MOF-NPs (MOF(Hb@AuNCs)-NPs). Scanning electron microscopy (B) and annular dark-field transmission electron microscopy (ADF-STEM) (C) images of MOF(Hb@AuNCs)-NPs. (D) ADF-STEM images of a MOF(Hb@AuNCs)-NP at different magnifications (i and ii). The AuNCs within MOF-NPs are highlighted with yellow dash circles. Energy dispersive X-ray elemental maps of Al (blue), Fe (red) and Au (yellow) of the same MOF(Hb@AuNCs)-NP(iii). The scale bars represent 20 nm. (E) UV-vis spectra of MOF(Hb@AuNCs)-NPs (i) and MOF(Hb)-NPs (ii) after purging with compressed air and nitrogen (N2) gas.
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