Metal-Organic Framework-Based Oxygen Carriers with Antioxidant Activity Resulting from the Incorporation of Gold-Based Nanozymes

doi:10.21203/rs.3.rs-1952010/v1

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Metal-Organic Framework-Based Oxygen Carriers with Antioxidant Activity Resulting from the Incorporation of Gold-Based Nanozymes

https://doi.org/10.21203/rs.3.rs-1952010/v1

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Blood transfusions are a life-saving procedure since they can preserve the body’s oxygen levels in patients suffering from acute trauma, undergoing surgery, receiving chemotherapy or affected by severe blood disorders. Due to the central role of haemoglobin (Hb) in oxygen transport, the so-called Hb-based oxygen carriers (HBOCs) are currently being developed for situations where donor blood is not available. In this context, an important challenge to be addressed is the oxidation of Hb into methaemoglobin (metHb) which is unable to bind and release oxygen. Since within red blood cells, this process is prevented by a set of protective enzymes such as superoxide dismutase and catalase, several research groups have considered incorporating these enzymes to create HBOCs with antioxidant properties. However, the use of biological enzymes has important limitations related to their high cost, potential immunogenicity or low stability in vivo. Thus, nanomaterials with enzyme-like properties (i.e., nanozymes (NZs)) have emerged as a promising alternative. In this work, we evaluate the antioxidant properties of gold (Au)-based NZs following incorporation within a type of HBOC previously reported by our group (i.e., Hb-loaded metal organic framework (MOF)-based nanocarriers (NCs)). We first prepare Au-loaded Au@MOF-NCs and demonstrate their ability to catalytically deplete two prominent reactive oxygen species (ROS) (i.e., hydrogen peroxide and superoxide radical) which exacerbate Hb’s autoxidation. Furthermore, these catalytic properties are retained over several cycles. Following loading with Hb, we show how the ROS-scavenging properties resulting from the Au-based NZs, translate into a decrease in metHb content. All in all, these results highlight the potential of NZs to create novel HBOCs with antioxidant protection which may find applications as a blood substitute in the future.

Antioxidant activity

Blood substitutes

Gold Nanoparticles

Haemoglobin-based Oxygen Carriers

Metal-organic Frameworks

Nanozymes

Transfusions of whole blood or packed red blood cells (RBCs) are a crucial procedure to restore tissue oxygenation in the context of massive trauma, to replenish blood loss during surgery, for the treatment of blood disorders such as anaemia and haemophilia, or for patients receiving chemotherapy. Despite being a widely used clinical procedure, blood transfusions have important limitations such as a limited supply, the need for blood group typing and matching due to the RBCs membrane antigens, the risk for disease transmission (i.e., HIV, malaria, hepatitis or more recently SARS-CoV-2), special storage requirements (i.e., they need to be kept in refrigerated conditions) or RBCs short shelf life (i.e., 42 days when stored in refrigerated conditions and only 1 day at room temperature (RT)). When traumatic injuries take place, the perishability and strict storage requirements of donor RBCs impede a facile and immediate (i.e., at the point-of-injury) administration. Hence, although time is absolutely critical, transfusion of RBCs takes place only after hospital admission.

Therefore, a great deal of research efforts is currently being put to develop RBCs surrogates that are free from the aforementioned drawbacks. Amongst the different strategies that are currently being pursued, the so-called haemoglobin-based oxygen carriers (HBOCs) are of particular interest. As indicated by their name, HBOCs make use of haemoglobin (Hb), which is the oxygen-carrying component of native RBCs. HBOCs overcome several limitations of donor RBCs since they have potentially unlimited availability, universal compatibility, can be prepared in large quantities and in sterile conditions and can also be stored at RT for extended periods of time.

Despite having optimal oxygen binding and releasing properties, free Hb cannot be administered into the body due to toxicity issues including renal toxicity, inflammation or vasoconstriction.^1,2 Thus, a main strategy to create successful HBOCs, relies on developing encapsulation platforms to preserve Hb’s structure and functionality. As such, Hb has already been entrapped within a myriad of systems which include liposomes, polymersomes or polymeric particles.^3–6 Among the reported Hb carriers, metal–organic framework (MOF) nanoparticles (NPs) are of particular interest. MOF-NPs have unique advantages for the encapsulation of fragile biomacromolecules which arise from their large surface areas and well-defined crystalline porous structures. We have recently reported the encapsulation of Hb within the so-called porous coordination network (PCN)-333(Al) which is a type MOF-NP constituted by Al3 + as the metallic ion and the organic linker 4,4’,4’’-s-triazine-2,4,6-triyl-tribenzoic acid (H₃TATB).^7,8 PCN-333 is one of the few water-stable MOF-NPs reported to date exhibiting large void volumes which allow for the high Hb loading that is required to achieve an oxygen-carrying capacity similar to that of blood. Additionally, PCN-333 displays one of the largest cages that make it possible to encapsulate individual Hb molecules within predefined cavities. That way Hb is protected from misfolding or denaturation thus preserving its excellent oxygen binding and releasing properties.

Despite the tremendous progress achieved in recent years, a challenge that still remains to be resolved is to prevent the oxidation of Hb into methaemoglobin (metHb). Due to the oxidized ferric state (Fe³⁺) of the heme iron, metHb is not able to carry and deliver oxygen and, furthermore, metHb conversion results in dysregulated vascular tone and inflammatory reactions.^9–11 Thus, native RBCs have the ability to prevent and revert this process by means of an antioxidant system involving prominent enzymes such as catalase (CAT), superoxide dismutase (SOD) and glucose oxidase. Inspired by this biological mechanism, several studies have reported the fabrication of HBOCs incorporating these antioxidant enzymes in an attempt to minimize metHb conversion.^12–14 However, important drawbacks including high cost, low stability and potential immunogenicity hamper the biomedical applications of enzymes, especially in the context of in vivo therapeutics.¹⁵ HBOCs with antioxidant coating layers have also been reported. Specifically, Hb molecules and also Hb-loaded micro- and nanoparticles have been coated with an antioxidant polydopamine (PDA) coating. We, and others, have showed the ability of such an approach to minimize the oxidation of the encapsulated Hb.^8,16−19 However, an important limitation of PDA is its uncontrollable disintegration in physiological conditions.^20–23 This is an important drawback for developing HBOCs that are expected to circulate in the vasculature for long periods of time. Another interesting concept is to make use of nanozymes (NZs), which are nanomaterials with catalytic properties that bypass some of the limitations of biological enzymes since they display high stability and are amenable for large scale production at a reasonable cost.^24,25 Furthermore, NZs experience better recyclability as compared to their biological counterparts and can sustain multiple rounds of catalytic activity. Being synthetic compounds, NZs are free from xenogeneic contamination and exhibit diminished batch-to-batch variations. HBOCs incorporating NZs include the work of Komatsu et al. which reported a Hb-albumin cluster incorporating platinum -NPs as the antioxidant NZs.²⁶ We recently reported poly(lactic-co-glycolic acid)-NPs decorated with Hb molecules and cerium oxide -NPs as the NZ component.²⁷ Both systems demonstrated the ability to reversibly bind and release oxygen and were also able to catalytically deplete two prominent reactive oxygen species (ROS) (i.e., hydrogen peroxide (H₂O₂) and superoxide radical anions (O₂^•−)) that exacerbate Hb’s oxidation into metHb. Considering these promising results, in this work, we further explore the use of antioxidant NZs for the design of successful HBOCs with decreased metHb conversion. We advanced our previously reported Hb-loaded PCN-333(Al) by incorporating NZs as an innovative tool to minimize the oxidation of the encapsulated Hb into metHb. For that, we assess the potential of gold (Au)-based NZs due to their tunability, biocompatibility and ease of synthesis at a relatively low cost.²⁸

2.1. Materials

Fresh bovine blood was obtained from SSI Diagnostica A/S (Hillerød, Denmark). Sodium chloride (NaCl), sodium hydroxide (NaOH), toluene, aluminium chloride hexahydrate (AlCl₃·6H₂O), N, N’-dimethylformamide (DMF), trifluoroacetic acid (TFA), acetone, ethanol, gold (III) chloride trihydrate (HAuCl₄·3H₂O), sodium borohydride (NaBH₄), polyvinyl pyrrolidone (PVP, 10 kDa), 4-(2-hydroxyethyl) piperazine-1-ethane-sulfonic acid (HEPES), hydrogen peroxide (H₂O₂), horseradish peroxidase (HRP), ethylenediaminetetraacetic acid (EDTA, 0.5 M) solution, cell proliferation reagent WST-1, xanthine, xanthine oxidase (XO) from bovine milk, Amicon Ultra-15 (30 kDa), Amicon Ultra-0.5 (100 kDa) filters and sodium dithionite (SDT) were obtained from Merck Life Science A/S (Søborg, Denmark). 4,4’,4’’-s-triazine-2,4,6-triyl-tribenzoic acid (H₃TATB) was obtained from ChemScene LLC (New Jersey, USA). Amplex red reagent, PrestoBlue cell viability reagent and Pierce bicinchoninic acid (BCA) protein assay kit were obtained from Thermo Fisher Scientific (Massachusetts, USA).

10 mM HEPES buffer was prepared with ultrapure water (Milli-Q (MQ), EMD Millipore, Massachusetts, USA) and the pH was adjusted to pH 7.4 by NaOH.

2.2. Hb Extraction from Bovine Blood

In this study, Hb was extracted from fresh bovine blood through a haemolysis treatment which was induced using a hypotonic solution as described in a previous study.²⁹ The fresh bovine blood was washed with an isotonic saline solution (0.9% NaCl, 3×, 15 min, 1500g) to collect RBCs. The obtained RBCs were lysed by mixing them with a hypotonic solution (MQ:toluene, 1:0.4, v/v) at a volume ratio of 1:1.4. Full lysis was achieved by thorough vortexing and, the lysed RBCs, were stored at 4°C overnight to allow for separation of the different phases using a separation funnel. The next day, three different layers were obtained with the bottom one containing the stroma-free Hb which was collected and spun down (20 min, 8000g). Next, the supernatant containing the stroma-free Hb was collected and filtered through ash-free filtration paper. The purified stroma-free Hb was stored at -80°C for future use.

2.3. Fabrication and Characterization of Au-based NZs

2.3.1. Synthesis of Au-based NZs. The reducing agent NaBH₄ (200 µL, 1 mg mL^− 1 in ice-cold MQ) was rapidly added to an aqueous solution of HAuCl₄·3H₂O (100 µL, 5.88 mM) which was diluted in 9.7 mL MQ. After magnetic agitation at the speed of 400 rpm for 5 min at 37°C, the reaction was stopped by spinning-down the unreacted reagents. The resulting bare Au-NPs were washed in MQ using Amicon Ultra-15 filters (30 kDa, 3×, 10 min, 5000g) and resuspended in 1 mL of MQ for future use. PVP-modified AuNPs were also fabricated and two different Au:PVP monomer molar ratios were considered: 1:5 (for Au/P5-NP) and 1:10 (for Au/P10-NP). For the preparation, a mixture of HAuCl₄·3H₂O (100 µL, 5.88 mM in MQ) and PVP (100 µL (for Au/P5-NP) and 200 µL (for Au/P10-NP) 3.27 mg mL^− 1) was diluted in MQ to a total volume of 10 mL and stirred for 30 min at 250 rpm and 37°C using a magnetic stirrer. Next, a NaBH₄ solution (200 µL, 1 mg mL^− 1 in ice-cold MQ) was rapidly added and further stirred for 5 min at 400 rpm and 37°C. Following several washes in MQ as previously described, the resulting PVP-modified Au-NPs (Au/P5-NP or Au/P10-NP) were collected and resuspended in 1 mL of MQ. The exact amounts of the different chemicals used for the preparation of the different NPs can be found in Table S1 (Supporting Information). All the prepared Au-based NZs were stored at 4 ℃ until future use.

2.3.2. Characterization of the Au-based NZs. The absorbance (Abs) spectra of the different NPs (i.e., Au-NP, Au/P5-NP and Au/P10-NP) were recorded in the wavelength range of 200–800 nm using a spectrophotometer (UV-2600 UV-Vis, Shimadzu Corp., Kyoto, Japan). The Zeta (ζ) -potentials of the different Au-based NZs were determined using a Zetasizer (nano-ZS, Malvern Panalytical Ltd., Malvern, UK) while the hydrodynamic diameter and polydispersity index (PDI) were assessed by dynamic light scattering (DLS) also using the Zetasizer. 2.3.3. Catalytic activity of Au-based NZs. 2.3.3.1. CAT-like activity. The CAT-like activity (i.e., H₂O₂ scavenging property) of both the bare and the PVP-stabilized Au-NPs was evaluated using the Amplex Red assay. In particular, 100 µL of the different Au-based NZs suspensions (i.e., Au-NP, Au/P5-NP or Au/P10-NP) at three different concentrations (i.e., 73.5 µM (low conc.), 147 µM (med. conc.) and 294 µM (high conc.)) were added to a mixture containing H₂O₂ (10 µL, 0.18 mM), HRP (100 µL, 2 U mL^− 1) and Amplex Red (10 µL, 0.1 mM) all in HEPES buffer using a black 96-well plate. The resulting suspensions were incubated for 1 h at RT and the fluorescence intensity (FI, λ_ex/λ_em = 530/586 nm) was recorded immediately after mixing and then every 5 min using a TECAN Spark multimode plate reader (Tecan Group Ltd., Maennendorf, Switzerland). HEPES buffer (100 µL) incubated with and without H₂O₂ (10 µL, 0.18 mM) were used as positive and negative controls, respectively.
2.3.3.2. SOD-like activity. The SOD-like activity (i.e., the ability to scavenge O₂^•−) of the different Au-based NZs was evaluated by the WST-1 assay. In particular, 100 µL of the different Au-based NZs suspensions (i.e., Au-NP, Au/P5-NP or Au/P10-NP) at three concentrations (i.e., 73.5 µM (low conc.), 147 µM (med. conc.) and 294 µM (high conc.)) were added to a mixture containing XO (10 µL, 0.05 U mL^− 1) and WST-1 reagent (100 µL, WST-1 (2.5% v/v), EDTA (0.1 mM) and xanthine (0.1 mM)) all in HEPES buffer using a transparent 96-well plate. The resulting suspensions were incubated for 1 h at RT and the Abs readings at 438 nm were recorded every 5 min using the plate reader. A solution of HEPES buffer (100 µL) and WST-1 (100 µL) with and without the addition of XO (10 µL) were used as positive and negative controls, respectively.

2.4. Fabrication and Characterization of MOF-Nanocarriers (NCs) Loaded with Au-based NZs

2.4.1. Fabrication of empty MOF-NCs. A mixture of AlCl₃·6H₂O (12 mL, 3 mg mL^− 1 in DMF), H₃TATB (12 mL, 1 mg mL − 1 in DMF) and TFA (40 µL) was allowed to react for 24 h at 95°C. The resulting MOF-NCs were then collected and washed in DMF (3×, 20 min, 15 000g), soaked (> 4 h each time) and washed in acetone (3×, 20 min, 15 000g) for solvent replacement and dried in a vacuum oven.

2.4.2. Fabrication of MOF-NCs loaded with Au-based NZs (Au@MOF-NCs). The Au@MOF-NCs were prepared using different Au inputs but keeping a constant molar ratio of 1:10 (Au: PVP monomer). Specifically, a mixture containing HAuCl₄·3H₂O (10, 25, 50 or 100 µL, 5.88 mM in MQ) and PVP (20, 50, 100 or 200 µL, 3.27 mg mL^− 1 in MQ) diluted in MQ to a final volume of 9 mL was stirred at the speed of 250 rpm for 30 min at 37°C. In parallel, 1 mL of the suspension of MOF-NCs (1 mg mL^− 1 in ice-cold MQ) was mixed with a NaBH₄ solution (20, 50, 100 or 200 µL, 1 mg mL^− 1 in ice-cold MQ) and incubated for 5 min on the ice bath. Next, this suspension was rapidly added to the HAuCl₄ and PVP mixture keeping a 1:10:10 molar ratio for Au:PVP monomer:NaBH₄. After incubation for 5 min at 400 rpm and 37°C and several washes in MQ (3×, 20 min, 15 000g), Au@MOF-NCs with increasing amounts of HAuCl₄· were obtained (i.e., Au10@MOF-NC, Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC when 10, 25, 50 and 100 µL of HAuCl₄·3H₂O 5.88 mM were used). The amounts and concentrations of the different chemicals used for the preparation of the different Au@MOF-NCs can be found in Table S1 (Supporting Information).

2.4.3. Characterization of MOF-NCs and Au@MOF-NCs. The ζ-potentials, hydrodynamic diameters and PDIs of the empty MOF-NCs and the different Au@MOF-NCs were assessed using the Zetasizer. Their morphology was evaluated using scanning electron microscopy (SEM, Thermo Fisher Scientific Teneo, MA, USA) at an accelerating voltage of 3 kV and a beam current of 13 pA. Scanning transmission electron microscopy (STEM) imaging and energy dispersive X-ray (EDX) spectroscopy were performed on a Thermo Fisher Scientific Tecnai-Osiris equipped with a high brightness Schottky X-FEG and Super-X EDX system comprising four silicon drift detectors and Bruker acquisition software (Bruker Daltonics Inc., MA, USA). Samples for STEM analysis were prepared by drop-casting the solution on a copper TEM grid with ultrathin carbon support. EDX data were collected in the form of spectrum images, in which a focused electron probe was scanned in a raster across a region of interest. For each scan point, structural information was obtained from the electron scattering incident on a high-angle annular dark-field detector (HAADF) and, simultaneously, an EDX spectrum was obtained by collecting X-rays emitted from the local volume probed by the electron beam. Spectrum images were acquired with a probe current of approximately 0.5 nA, and a beam energy of 200 keV.

2.4.4. Catalytic properties of MOF-NCs and Au@MOF-NCs. 2.4.4.1. CAT-like activity. The ability of the Au@MOF-NCs to scavenge H₂O₂ was evaluated using the Amplex Red assay in a similar way as described in section 2.3.3.1. Briefly, 200 µL of the different Au@MOF-NCs suspensions (i.e., Au10@MOF-NC, Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC) at a concentration of 0.5 mg mL^− 1 were mixed with H₂O₂ (10 µL, 0.18 mM), all in HEPES buffer, and incubated for different time intervals (i.e., 15, 30 and 60 min) using a thermoshaker (1200 rpm, 37 ℃, HMT Thermoshaker, Grant-bio, UK). Next, the samples were spun down (5 min, 15 000g) and corresponding supernatants (180 µL) were collected and mixed with HRP (100 µL, 2 U mL^− 1) and Amplex Red (10 µL, 0.1 mM), all in HEPES buffer, followed by 5 min incubation in the thermoshaker (1200 rpm, 37 ℃). Subsequently, the supernatants (180 µL) were transferred to a black 96-well plate for FI measurements (λ_ex/λ_em = 530/586 nm) using the plate reader. HEPES buffer (200 µL) incubated with and without H₂O₂ (10 µL, 0.18 mM) were used as positive and negative controls, respectively. The normalized mean FI (nMFI) was calculated as follows: % nMFI = (MFI of sample - MFI of negative control)/ (MFI of positive control - MFI of negative control) × 100.

Multiple rounds of CAT-like activity were also evaluated. To this end, after a first round of catalytic activity for 30 min, the different suspensions of Au@MOF-NCs (200 µL, 0.5 mg mL^− 1) were spun down (5 min, 15 000g) and washed in HEPES buffer (1×, 5 min, 15 000g). Next, the samples were incubated again with a fresh H₂O₂ solution to conduct a second round of scavenging reaction. The procedure was repeated for a total of 5 cycles and the nMFI was calculated as previously described. At least two independent experiments were conducted for each condition.

2.4.4.2. SOD-like activity. The ability of the different Au@MOF-NCs to scavenge O₂^•− was evaluated using the WST-1 assay as shown in section 2.3.3.2. In particular, 200 µL of the different Au@MOF-NCs suspensions (i.e., Au10@MOF-NC, Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC) at a concentration of 0.5 mg mL^− 1, were mixed with XO (10 µL, 0.05 U mL^− 1) and a WST-1 reagent solution (200 µL containing WST-1 (2.5% v/v), EDTA (0.1 mM) and xanthine (0.1 mM)), all in HEPES buffer. The suspensions were incubated for different time intervals (i.e., 15, 30 and 60 min) in the thermoshaker (1200 rpm, 37°C). After spinning them down (5 min, 15 000g), the supernatants were collected (180 µL) and transferred to a 96-well plate and the Abs was recorded at 438 nm using the plate reader. A mixture of HEPES buffer (200 µL) and WST-1 (200 µL) solution with and without XO (10 µL) were used as positive and negative controls, respectively. The normalized Abs (nAbs) was calculated as follows: % nAbs = ((Abs of sample - Abs of negative control)/(Abs of positive control - Abs of negative control)) × 100.

Multiple rounds of SOD-like activity for the different Au@MOF-NCs was also evaluated. For that, Amicon Ultra-0.5 filters (100 kDa) were used as reaction containers since this allows to remove the unreacted xanthine, EDTA and WST-1 reagents as filtrates by spinning-down the reaction mixture. Although XO, due to its large size (270 kDa), cannot be removed using the Amicon Ultra-0.5 filters, its influence can be neglected as we showed in our previous work.30 Thus, after the first round of catalytic activity for 30 min, the suspensions were spun down (5 min, 15 000g), the filtrates were collected (180 µL) and the Abs readings were recorded. Next, the Au@MOF-NCs collected in the filter, were washed with HEPES buffer (2×, 5 min, 15 000g) and incubated again with fresh xanthine, XO and WST-1 solutions, conducting a second round of O₂^•− scavenging. This process was repeated for a total of five cycles and the nAbs was calculated as previously described. At least two independent experiments were carried out.

2.5. Fabrication and Characterization of Hb-loaded Au@MOF-NCs (Au@MOF^Hb-NCs).

2.5.1. Hb loading. For Hb encapsulation, bovine Hb (20 µL, 100 mg mL^− 1 in MQ) was added to suspensions of either MOF-NC or Au@MOF-NCs (1 mL, 1 mg mL^− 1 in MQ) under continuous magnetic stirring at 800 rpm and at RT for 2 h. The resulting Hb-loaded MOF-NC (i.e., MOF^Hb-NC) or Au@MOF-NCs (i.e., Au10@MOF^Hb-NC, Au25@MOF^Hb-NC, Au50@MOF^Hb-NC and Au100@MOF^Hb-NC) were obtained after washing with MQ (3×, 20 min, 15 000g).
2.5.2. Characterization of Au@MOF ^Hb -NCs. The ζ-potentials, hydrodynamic diameters and PDIs of the Hb-loaded Au@MOF^Hb-NCs fabricated with different Au inputs were assessed using the Zetasizer. Their morphology was evaluated using both SEM and STEM as described in section 2.4.3.
2.5.3. Assessment of Hb’s structure. The secondary structure of Hb was assessed by Fourier-transform infrared spectroscopy (FTIR) using a FTIR spectrometer (PerkinElmer Spectrum 100, PerkinElmer Inc., Massachusetts, USA) with a pre-installed universal attenuated total reflectance component. For FTIR analysis, free Hb, MOFHb-NP and the different Au@MOF^Hb-NCs (i.e., Au10@MOF^Hb-NC, Au25@MOF^Hb-NC, Au50@MOF^Hb-NC and Au100@MOF^Hb-NC) were freeze-dried to obtain powdered products. The spectra were collected within the wavelength range of 800 to 2000 cm^− 1 with a resolution of 4 cm^− 1 and four scans per sample were recorded. The second derivative of the Amide I region was conducted using the PeakFit software. To get information about Hb’s secondary structure, the spectra in Amide I region were further analysed using the pre-installed AutoFit function by Gaussian deconvolution fitting (version 4.12, SeaSolve Software. Inc., CA, USA).
2.5.4. Binding and releasing of molecular oxygen (O ₂ ). The O₂ binding/releasing ability of Hb after loading within the final product was evaluated by UV-vis spectroscopy. Herein, the Au50@MOF^Hb-NC was selected and suspended in HEPES buffer at the Hb concentration of 0.1 mg mL^− 1, and the UV-vis spectra (350–650 nm) were recorded using the UV-vis spectrophotometer. Then, the suspension was purged with N₂ flow together with a pinch of oxygen scavenger SDT for 10 min to obtain deoxygenated Hb (deoxy-Hb), followed by recording its UV-vis spectrum. Next, the suspension was purged with air flow for 10 min to obtain oxygenated Hb (oxy-Hb) and its UV-vis spectrum was recorded. Subsequently, the suspension of Au50@MOF^Hb-NC was purged with N₂ and air flow again and their UV-vis spectra were recorded correspondingly.

2.5.5. CAT-like activity of Au@MOF ^Hb -NCs. The ability of the final product Au@MOF^Hb-NCs to scavenge H₂O₂ was evaluated using the Amplex Red assay as described in the section of 2.4.4.
2.5.6. Antioxidant protection. The change in Soret peak height of free Hb and Hb encapsulated within MOF^Hb-NC and the different Au@MOF^Hb-NCs (i.e., Au10@MOF^Hb-NC, Au25@MOF^Hb-NC, Au50@MOF^Hb-NC and Au100@MOF^Hb-NC) was recorded using the UV-vis spectrophotometer. For that, suspensions of free Hb, MOF^Hb-NC or the different Au@MOF^Hb-NCs (2 mL, 0.1 mg mL^− 1 in Hb concentration) were mixed with a solution of H₂O₂ (20 µL, 100 mM), all in HEPES buffer, and the Soret peak was recorded following incubation for 15 min.

3.1. Fabrication and Characterization of Au-based NZs.

Au-based NZs were fabricated using a one-pot method following a previously reported protocol. Since several studies have demonstrated that the interaction of Au-NPs with polymers has a strong and positive impact on their size, distribution and stability, bare Au-NP but also PVP-stabilized Au/P-NPs were considered (Fig. 1A).³¹ For the PVP-stabilized Au/P-NPs, two different molar ratios of Au:PVP monomer were evaluated (i.e., a 1:5 molar ratio for Au/P5-NP and a 1:10 molar ratio for Au/P10-NP). The UV-vis spectra indicate the formation of both bare and PVP-stabilized Au/P-NPs as shown by the presence of Abs bands in the visible spectral region which are known as surface plasmon resonance (SPR) (Fig. 1B).^32–34 The localized SPR centred at ~ 520 nm suggests the formation of spherical PVP-stabilized Au/P-NPs while the red shift and the broadening of the SPR band arising from the bare Au-NPs suggests potential aggregation of the NPs when PVP was not present.³¹ This result confirms the stabilization effect of PVP thanks to the repulsive forces arising from its hydrophobic carbon chains and to the oxygens of the carbonyl groups which are able to hydrogen bond with water.³⁵ The presence of the PVP-coating was also demonstrated by the high Abs reading at ~ 200 nm, which could be attributed to the presence of carbonyl functional groups in the ring structure (n→π* transition) of PVP. In addition, the presence of PVP was further verified by ζ-potential measurements. Figure 1C shows the expected reduction in negative ζ-potential (i.e., from − 27.7 ± 1.4 mV for the Au-NP to -6.3 ± 3.8 and − 8.0 ± 1.5 mV for Au/P5-NP and Au/P10-NP, respectively) since a PVP coating will hinder the negative charge of the underlying Au-NP. The stabilization effect rendered by PVP was further evaluated by freeze-drying the different Au-based NZs and resuspending them again in MQ. The size of bare Au-NP increased from ~ 6 to ~ 66 nm while only an increase of ~ 5 and ~ 4 nm in size was observed for Au/P5-NP and Au/P10-NP, respectively (Figure S1, Supporting Information). Furthermore, the size change of freshly prepared Au-based NZs after 30 days of storage at 4 ℃ was also determined. Figure 1D shows how both the bare Au-NP and the PVP-stabilized Au/P-NPs display a similar hydrodynamic diameter of ~ 5–6 nm and a PDI ~ 0.2–0.3 when freshly prepared (day 1). However, the results are different following storage for 30 days and, while a slight size increase of ~ 2–3 nm is observed for the PVP-stabilized Au/P-NPs, the uncoated Au-NP experience a large increase in both size (to ~ 15 nm) and PDI (up to 0.8) indicating aggregation of the sample. The aggregation of the bare Au-NP is further confirmed by the change of the colour of the suspension (from red to blue) resulting from the interparticle plasmon coupling (Fig. 1E).³¹

3.2. Catalytic Activity of the Au-based NZs

ROS such as H₂O₂ or O₂^•− promote and enhance the oxidation of Hb into non-functional metHb. While native RBCs can avoid Hb’s autoxidation making use of several enzymes including CAT, SOD or glucose oxidase, in this study we make use of NZs.^12–14 NZs circumvent important drawbacks of biological enzymes which include high cost, low stability and immunological reactions.¹⁵ NZs, which are nanomaterials with catalytic properties, exhibit high stability and can be produced in large quantities at low cost.^24,25 Amongst the different NZs with reported antioxidant activity, Au-based NZs are of particular interest due to their tunability, biocompatibility and ease of synthesis.²⁸ Thus, the aim of this study is to evaluate whether the antioxidant activity of the as-synthetized Au-based NZs translated into decreased metHb conversion following incorporation within a HBOC.

Hence, as a first step, we explored the CAT- and SOD-like activity of the different Au-based NZs formulations (i.e., bare Au-NP and PVP-stabilized Au/P5-NP and Au/P10-NP), that is, their ability to scavenge H₂O₂ or O₂^•−. Specifically, the CAT-like activity was assessed by the Amplex Red assay. With this assay, H₂O₂ in the presence of the enzyme HRP, acts as a co-substrate for the oxidation of the Amplex Red probe into the fluorescent resorufin product. Thus, compounds that display CAT-like activity and are therefore able to catalyse the disproportionation of H₂O₂ into H₂O and O₂, result in decreased production of the fluorescent resorufin product. Figure 2A shows the FI readings of bare Au-NP (Fig. 2Ai) and the PVP-coated Au/P5-NP (Fig. 2Aii) and Au/P10-NP (Fig. 2Aiii) at three different concentrations (i.e., low, med. and high conc.). The three different formulations at the three studied concentrations display CAT-like activity as shown by the decrease in FI as compared to the positive control. For the bare Au-NP at low conc., a ~ 3500 decrease in FI as compared to the positive control can be observed at the first FI reading time point (i.e., 1 min), while a reduction of ~ 5800 and ~ 9600 are observed for the Au-NP at the med. and high conc., respectively. This suggests that expected concentration-dependent decreases in FI. For longer time points, the signal from the Au-NP at high conc. remains stable, suggesting that the H₂O₂ scavenging reaction had been completed. However, the results were different from Au-NP at the low and med. conc. where the FI signal continued to decrease over time which we attribute to photobleaching caused by the degradation of the resorufin fluorophore by H₂O₂.^36,37 We speculate that, due to the enhanced activity of Au-NP at high conc. as compared to the low or med. conc., less fluorophore and H₂O₂ remain available for photobleaching or degradation. In contrast, the PVP-coated counterparts display a stronger and concentration-dependent CAT-like activity and no degradation of the resorufin fluorophore can be detected over time. In particular, for both Au/P-NPs at the lowest studied concentration, a ~ 10 800 and a ~ 10 300 decrease in FI is observed for Au/P5-NP and Au/P10-NP, respectively. Increasing the concentrations resulted on a further decrease in FI for the two PVP-coated formulations, indicating the expected higher scavenging of H₂O₂ when increased amounts of NPs are used. At the beginning of reaction, a ~ 19 700 and a ~ 18 600 decrease in FI as compared to the positive control is detected for Au/P5-NP and Au/P10-NP, respectively. Such enhanced catalytic activity following incorporation of a PVP coating is not surprising; since the chemical reactions take place at the interfacial perimeter around the NPs, any surface coating layers will have an effect on the catalytic properties.³⁸ The SOD-like activity of the Au-based NZs, that is, their ability to scavenge O₂^•−, was evaluated next and, for that, the WST-1 assay was employed (Fig. 2B). The method relies on the ability of xanthine and the XO enzyme to generate O₂^•− from O₂. Next, the WST-1 tetrazolium salt is reduced by O₂^•− to formazan which can be detected by its characteristic Abs peak at 450 nm. Thus, compounds with the ability to scavenge of O₂^•− will reduce the production of WST-1 formazan and the subsequent decrease in Abs readings. Figure 2B shows how the bare and the PVP-coated Au-based NZs display SOD-like activity as shown by the decrease in Abs as compared to the positive control. The reaction was completed after 35 min and, in this case, the O₂^•− scavenging properties are concentration-dependent for the three different formulations. Interestingly, enhanced SOD-like activity is observed for the PVP-coated Au/P-NPs as compared to the bare Au-NP and this is more obvious for the lowest concentration. Specifically, the bare Au-NP after 35 min of reaction only promote a ~ 0.24 decrease in Abs while the decrease in Abs is of ~ 0.35 for both Au/P5-NP and Au/P10-NP at the same studied condition (i.e., after 35 min of reaction and for the lowest studied concentration).

3.3. Fabrication and Characterization of Au@MOF-NCs

We recently presented a novel HBOC consisting of Hb-loaded MOF-based NPs. Specifically, we employed the so-called PCN-333(Al) MOF which is constituted by trivalent metal species (i.e., Al³⁺) and the organic linker H₃TATB. Important features of PCN-333(Al) making it a very attractive platform to create HBOCs, is its stability in aqueous media and the fact that it displays one of the highest void volumes and largest cages amongst the different MOFs reported to date.^7,8 While the high void volume allows the encapsulation of large amounts of Hb which is important to meet the high oxygen demands of our body, the large cages enable the encapsulation of single Hb molecules within individual cages. This is an important feature to minimize Hb’s aggregation and leaching. Thus, nano-sized MOF-NCs with a PCN-333(Al) crystalline structure were prepared and, prior to Hb loading, the Au-based NZs were embedded by the in situ reduction of HAuCl₄ (Fig. 3A). Due to superior catalytic properties of Au/P-NPs as compared to the bare Au-NPs counterparts, PVP was used as a stabilizer. The resulting Au@MOF-NCs were prepared with increasing amounts of HAuCl₄ but keeping a constant Au:PVP monomer molar ratio of 1:10. Specifically, Au10@MOF-NC, Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC were obtained when 10, 25, 50 and 100 µL, respectively, of a 5.88 mM HAuCl₄ solution were added to the mixture. Figure 3B depicts photographic images of empty MOF-NC and the different Au@MOF-NCs. While the suspension of empty MOF-NC shows a white milky colour, different Au@MOF-NCs look rather pinkish suggesting formation of Au-based NZs within the MOFs. Furthermore, the higher the Au input used for their assembly, the stronger the colour of the suspension. Figure 3C shows the ζ-potentials of the different formulations where, the empty MOF-NC, display the expected negative ζ-potential of -21.7 ± 0.1 mV which is attributed to the unreacted carboxylic acid groups of H₃TATB. In contrast, the in situ reduction of the HAuCl₄ solution promoted a decrease in negative ζ-potential suggesting the incorporation of PVP-stabilized Au/P-NPs. Since both Au/P5-NP and Au/P10-NP exhibit almost neutral ζ-potentials of -6.3 ± 3.8 mV and − 8.0 ± 1.5 mV, respectively, (see Fig. 1C for details), it is expected that their incorporation within MOF-NCs renders Au@MOF-NCs with less negative ζ-potential values. In agreement with Fig. 1A, increasing Au inputs results in less negative ζ-potentials suggesting that higher amounts of HAuCl₄, result in enhanced incorporation of Au/P10-NP within the Au@MOF-NC. The hydrodynamic diameter was similar for empty MOF-NC and the different Au@MOF-NCs indicating good dispersibility (Fig. 3D). The PDI values for all the formulations are below 0.2 which makes them suitable for pharmaceutical applications.³⁹ The empty MOF-NC and different Au@MOF-NCs were characterized by SEM and STEM. Figure 4A shows SEM micrographs of MOF-NC and Au50@MOF-NC indicating that both types of NCs are monodisperse in size and display a close-to-spherical morphology. The structure of MOF-NC and Au50@MOF-NC were further explored by STEM-EDX (Fig. 4B). The magnified STEM image and its Fast Fourier Transform (FFT) image (see inset) suggest that the MOF-NC have a pore size of around 6.8 nm which make it suitable for Hb (6 × 6 × 5 nm) encapsulation. The STEM images and EDX elemental maps of Au50@MOF-NC confirm that the Au/P10-NP (~ 3 nm) were successfully incorporated into MOF-NC and distributed homogenously within Au50@MOF-NC (Fig. 4Biii and Fig. 4Biv).

3.4. Catalytic Activity of the Au@MOF-NCs

We next evaluated whether the incorporation of the Au-based NZs translated into CAT- and SOD-like activity for the resulting Au@MOF-NCs. Figure 5Ai shows the nMFI readings arising from the fluorescent resorufin product for Au@MOF-NCs fabricated with different Au inputs. CAT-like activity was already observed for Au@MOF-NCs assembled with the lowest Au input (i.e., Au10@MOF-NC) where, a ~ 15% decrease in nMFI, could be detected for the three studied time points, suggesting that the reaction was already completed after 15 min. Au@MOF-NCs fabricated with a higher Au input (i.e., Au25@MOF-NC) promoted an additional ~ 20% decrease in nMFI. Although further increases in the Au inputs did not promote an additional decrease in nMFI, these results suggest the incorporation of Au/P10-NP with preserved functionality.

Next, we assessed whether the different Au@MOF-NCs were able to conduct several cycles of H₂O₂-scavenging activity. Since HBOCs are expected to remain in the bloodstream for long periods of time, it is also important that they retain the ability to deplete ROS over multiple rounds. For that, we conducted the H₂O₂-scavenging reaction over five subsequent cycles by exchanging the resorufin product by a solution containing fresh Amplex Red and H₂O₂ (Fig. 5Aii). For the Au@MOF-NCs fabricated with the lowest Au input (i.e., Au10@MOF-NC), a ~ 25% decrease in nMFI is observed for the first cycle and such a decrease is reduced to only ~ 12 and ~ 5% for the second and fourth cycles, respectively. This indicates that the slight catalytic properties towards the decomposition of H₂O₂ of the Au10@MOF-NC are almost completely lost after five cycles of reaction. For the Au@MOF-NC assembled with higher amounts of Au (i.e., Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC) the results are different. Independently of the Au input, the Au@MOF-NCs were able to deplete ~ 45% nMFI after the first cycle of reaction and, each subsequent round, resulted in only a ~ 10% decrease on their scavenging ability. That is, there was a decrease in nMFI of ~ 35, 25 and 15% after two, three and four cycles or reaction. For the Au@MOF-NCs fabricated with a 100 Au input, the results were slightly different and, 25% of H₂O₂-scavenging properties, remained after four rounds of activity. What is more, in the fifth cycle, no further decrease in nMFI was detected for any of the Au@MOF-NCs. This suggests that the as-prepared MOF-NCs loaded with different amounts of Au-based NZs, are a robust system displaying up to 25% of the H₂O₂-scavenging activity even after several rounds of spinning and resuspension.

The SOD-like activity of the Au@MOF-NCs assembled with different Au inputs was evaluated by the WST-1 assay. Figure 5Bi shows the nAbs readings after 15, 30 and 60 min of incubation. For the Au@MOF-NCs fabricated with the lowest Au input (i.e., Au10@MOF-NC), a ~ 22 and a ~ 30% decrease in nAbs could be detected after 15 and 60 min of reaction, respectively. This indicates that such a low Au input was already enough to promote both H₂O₂ and O₂^•− scavenging properties. Upon increasing the Au input, a further decrease in nAbs was observed suggesting concentration-dependent O₂^•− scavenging properties. Specifically, for the longest incubation period, a ~ 40, ~52 and ~ 60% decrease in nAbs was detected for Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC, respectively. Multiple rounds of O₂^•− scavenging were also considered. Figure 5Bii shows how Au@MOF-NCs prepared with low Au inputs (i.e., Au10@MOF-NC and Au25@MOF-NC) experienced a substantial loss in scavenging activity already during the second cycle as shown by the ~ 25% decrease in nAbs. Additionally, only ~ 10% of O₂^•− scavenging properties remained after the fifth cycle as demonstrated by the ~ 10% decrease in nAbs. In contrast, the recyclability proved to be better for the Au@MOF-NCs prepared with the highest Au inputs (i.e., Au50@MOF-NC and Au100@MOF-NC). In this case, while a ~ 50% decrease in nAbs was detected after the second cycle, no further decrease could be observed after the third and fourth cycles, respectively. Importantly, even after five rounds of reaction, both Au50@MOF-NC and Au100@MOF-NC still preserve ~ 40% of their O₂^•− scavenging activity, again highlighting the robustness of such a system.

3.5. Fabrication and Characterization of Au@MOF^Hb-NCs.

Hb was incorporated within the Au@MOF-NCs fabricated with different Au inputs via post-encapsulation and, for that, we followed our previously reported approach.^7,8 Instead of human Hb, in this work we also chose to encapsulate bovine Hb (Fig. 6A). While human Hb requires the presence of 2,3-diphosphoglycerate as allosteric effector, bovine Hb depends only on chloride ions for native operation which are already present in blood and can also be provided by commonly used saline solution.⁴⁰ The loading of Hb within empty MOF-NC and the different Au@MOF-NCs (i.e., Au10@MOF-NC, Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC) was assessed by ζ-potential (Fig. 6B). While, as shown in Fig. 3B, both empty MOF-NC and the Au@MOF-NCs displayed negative ζ-potentials, a charge reversal was observed following incubation of the NCs with Hb which points towards its successful encapsulation. The observed positive ζ-potentials are a result of Hb’s positive charge at pH values that are lower than its isoelectric point (i.e., the isoelectric point of Hb is 6.8 and the pH of the MOF-NC and Au@MOF-NCs is ~ 5.6).⁷ These changes in ζ-potential are lower the higher the Au input used for the preparation of the Au@MOF-NCs, which suggests that the presence of Au-based NZs within the NCs, restricts the amount of encapsulated Hb. As such, a change in ζ-potential of ~ 32 mV was observed upon loading Hb into empty MOF-NCs (i.e., from − 21.7 mV to + 10.5 mV) while the change in ζ-potential after the incorporation of Hb within Au10@MOF-NC is of only ~ 26 mV. These changes in ζ-potentials are even smaller when higher Au inputs are used since, only a change of ~ 23, ~20 and ~ 14 mV, was detected for Au25@MOF-NC, Au50@MOF-NC and Au100@MOF-NC, respectively. Figure 6C shows the size and PDI of the MOF-NC and the Au@MOF-NCs before and after loading with Hb. Upon Hb encapsulation, an increase in both size and PDI can be observed for all the formulations, and such an increase is higher for the Au@MOF^Hb-NCs fabricated with the highest Au inputs. Thus, an increase of ~ 88 nm is observed for MOF^Hb-NC as compared to empty MOF-NC while the increase in size is of ~ 179 nm for the Au100@MOF^Hb-NC as compared to Au100@MOF-NC. The morphology of the Au50@MOF^Hb-NC was imaged both by SEM and STEM. The SEM micrograph shows preservation of the spherical morphology (Fig. 6Di) while the STEM image further confirms the incorporation of Au-based NPs within the Hb-loaded MOF structure. This was also verified by EDX elemental map showing how the regions with higher signal in the HAADF-STEM (mass-thickness contrast) image in Fig. 6Dii overlapped well with the EDX Au signal in Fig. 6Diii. The EDX elemental maps also show that the Al and Fe elements are uniformly distributed in the MOF material. Al is the element constituting the nodes of the MOF-NC, and Fe arises from the heme groups of Hb, indicating the Hb molecules distributed homogenously in the MOF materials (Fig. 6Diii).

Since protein structure is crucial to its function, before assessing Hb’s functionality within Au@MOF^Hb-NCs, we monitored any potential changes in its structure by FTIR spectroscopy. Figure 7Ai shows the comparison among the FTIR spectra of free Hb, empty MOF-NC and the different Hb-loaded samples. Empty MOF-NC show the expected bands of the H₃TATB linker, that is as phenyl ring (i.e., 1450 cm^− 1, stretching of phenyl ring; 1018 cm^− 1, in-plane C-H bending of phenyl ring) and triazine ring (i.e., 1357 cm^− 1, semicircle stretching of triazine ring; 1520 cm^− 1, quadrant stretching of triazine ring). The spectrum of free Hb exhibited the prominent characteristic peaks associated with Hb, such as amide I (i.e., 1652 cm^− 1, C = O stretching vibrations and out of plane C − N stretching), amide II (i.e., 1536 cm^− 1, N − H bending and C-N stretching). Following encapsulation of Hb within the different MOF-NCs (i.e., MOF^Hb-NC and the different Au@MOF^Hb-NCs), the signals arising from the MOF structure overlapped with the amide II region while no apparent modification was observed in the amide I region (Fig. 7Ai). Thus, the amide I bands (i.e., 1600–1700 cm^− 1), which are also the most prominent and sensitive region of the backbone of the protein, were selected for further analysis of Hb’s structure.^41–43 Fig. 7Bi shows the second derivatives of the FTIR spectra of the amide I region which were conducted with the goal to analyse these changes in a quantitative manner. For free Hb (red line), a peak around 1655 cm^− 1 was observed, which could be attributed to the α-helices, while a shoulder located in the range of 1620–1640 cm^− 1 could be associated with β-sheets. The peak around 1685 cm^− 1 could be attributed to β-turn structures. After loading into the MOF structures (i.e., MOF^Hb-NC and Au@MOF^Hb-NCs), the main peaks associated with Hb’s α-helices (i.e., at 1655 cm^− 1) and β-turns (i.e., at 1685 cm^− 1) were well preserved. However, the peaks associated with Hb’s β-sheets (i.e., the shoulder around 1620–1640 cm^− 1) were difficult to distinguish. Thus, further quantitative analyses were conducted, and the secondary derivatives of amide I spectra were deconvoluted and then fitted with a Gaussian multi-peak fitting function using the Peakfit software (version 4.12) to elucidate the secondary structure. Figure 7Bii shows the deconvoluted spectra of free Hb where the contribution of the different structures to the overall conformation can be observed. Specifically, six secondary conformational bands were revealed, including intermolecular aggregates (~ 1618 cm^− 1), β-sheets (~ 1631 cm^− 1), random coils (~ 1644 cm^− 1), α-helices (~ 1655 cm^− 1), β-turn structures (~ 1668 cm^− 1) and intramolecular aggregates (~ 1682 cm^− 1).^44–47 The fitted peak is in good agreement between the original (i.e., observed peak) and deconvoluted peaks (i.e., predicted peak). Figure 7C shows the contributions of the different structural elements for free Hb, MOF^Hb-NC and the different Au@MOF^Hb-NCs, which have been calculated as the percentage of the area of each peak to the total amide I area. The results show how, incorporation of Hb within the MOF^Hb-NC, promotes a slight decrease in β-sheets accompanied by a slight increase in the α-helix content. For Au@MOF^Hb-NCs, the results are different and depend on the Au inputs. Thus, for the lowest Au input (i.e., Au10@MOF^Hb-NC) no apparent change in the contribution of the different structural elements can be observed. However, for Au inputs of 25 and 50, the encapsulated Hb experiences an increase in the amount of β-sheet content and a decrease of β-turns. The biggest changes in the composition of structural elements are experienced for Hb entrapped within Au100@MOF^Hb-NC, where the β-sheet content decreases from 17.7% for free Hb to 7.2%. This decrease is accompanied by an increase in both β-turn (from 14.6% for free Hb to 19.7%) and α-helix content (from 26.7% for free Hb to 31.7%).

We next evaluated whether these changes in secondary structure had an effect on Hb’s ability to bind and release oxygen since, preserving Hb’s functionality, is arguably the most important feature for HBOCs. Figure 8 shows following purging with compressed air, the Hb entrapped within the Au50@MOF^Hb-NC, which were selected as a representative sample, displays the characteristic Abs bands of oxy-Hb. That is, a main band at 414 nm (known as the Soret peak) and two additional bands in the visible region at 543 and 577 nm (known as the Q-bands). Following purging with N₂, the Soret peak shifted to 430 nm, and a single band appeared at 566 nm, indicating the presence of deoxy-Hb. Following on, the Au50@MOF^Hb-NC were purged with air, N₂, and again with air and the corresponding shifts to oxy- and deoxy-Hb were observed for each cycle. Thus, importantly, these results highlight preservation of Hb’s structure following encapsulation within a MOF-NC containing Au-based NZs.

3.6. CAT-like activity of Au@MOF^Hb-NCs and Antioxidant Protection towards Conversion into metHb

Following Hb encapsulation, we assessed the catalytic antioxidant activity of the resulting Au@MOF^Hb-NCs and evaluated whether these properties translated into decreased metHb conversion. Figure 9A shows the nMFI resulting from the fluorescent resorufin product for MOFHb-NC and the different Au@MOF^Hb-NCs (i.e., Au10@MOF^Hb-NC, Au25@MOF^Hb-NC, Au50@MOF^Hb-NC and Au100@MOF^Hb-NC). MOF^Hb-NC promote a very pronounced decrease of ~ 93% in nMFI for the three studied time intervals. The results are different for the Au@MOF^Hb-NCs. Both Au10@MOF^Hb-NC and Au25@MOF^Hb-NC promote a decrease in nMFI of only ~ 45–50%. We hypothesize that the Au-based NZs incorporated within the NCs are able to slow down the H₂O₂–scavenging by the encapsulated Hb. Increasing the amounts of Au-based NZs does not result on a further decrease in H₂O₂–scavenging. On the contrary, a sharp decrease of ~ 80% and ~ 60% is observed for Au50@MOF^Hb-NC and Au100@MOF^Hb-NC, respectively. Although we cannot explain yet this different behaviour for the Au@MOF^Hb-NCs fabricated with the highest Au inputs, such a decrease in nMFI is still lower than the one observed for MOF^Hb-NC suggesting some level of antioxidant protection rendered by the in situ formed Au-based NZs.

Considering these encouraging results, we next evaluated if the slower H₂O₂-scavenging rate translated into decreased metHb conversion. To this aim, we monitored the decrease in the height of the characteristic Soret peak of oxy-Hb after incubating the MOF^Hb-NC and the different Au@MOF^Hb-NCs with H₂O₂ for 15 min. H₂O₂ was added to the suspension to accelerate Hb’s oxidation into metHb. Figure 9B shows that, although very slight, the incorporation of Au-based NZs within the NCs results in decreased metHb conversion. Specifically, while MOF^Hb-NC promote a ~ 18% decrease in the Soret peak, the decrease for the different Au@MOF^Hb-NCs is only of ~ 10%. Such antioxidant protection is independent of the Au input used for the fabrication of the different Au@MOF^Hb-NCs.

We recently reported the fabrication of a novel HBOCs consisting of Hb-loaded PCN-333(Al)-NPs which are a type of MOF-NPs displaying one of the highest void volumes and largest cages reported to date. The ability to incorporate large amounts of Hb which can be entrapped within individual cages, making PCN-333(Al)-NPs a powerful platform to create RBC surrogates. However, a long-standing challenge to create successful HBOCs lies in avoiding or at least minimizing Hb’s autoxidation into non-functional metHb. While, in our previous work, we employed an antioxidant PDA coating to fabricate Hb-loaded PCN-333(Al)-NPs with diminished metHb conversion, an important limitation of such an approach is the lack of stability of PDA in physiological conditions.^20,21 Thus, in this work, antioxidant NZs are explored as a potential solution to avoid Hb’s autoxidation. Specifically, Au-based NZs are fabricated with and without PVP as a stabilizer and two different formulations are considered. The resulting Au-based NZs are characterized in terms of size, PDI and catalytic properties towards the depletion of two prominent ROS (i.e., H₂O₂ and O₂^•−). The optimized conditions are used for the in situ formation of Au-based NZs within the MOF-NCs. Four different Au@MOF-NCs are successfully fabricated employing different Au inputs as shown by STEM and EDX analyses. Preservation of the catalytic activity of the entrapped Au-based NZs is shown by their ability to deplete H₂O₂ and O₂^•− also over multiple rounds. Finally, Hb is entrapped within the Au@MOF-NCs by post-encapsulation. Preservation of Hb’s ability to bind and release oxygen is demonstrated. The resulting Au@MOF^Hb-NCs are also able to deplete H₂O₂ and this results in a slight decrease in metHb conversion.

AUTHOR CONTRIBUTIONS

X.L. and L.H-R. conceived the study and were in charge of overall direction and planning. X.L. carried out the experiments. N.P.D. and E.O. performed the STEM, EDX and HAADF characterisation. X.L. and L.H-R. carried out the interpretation of the results. L.H-R. took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript.

AUTHOR INFORMATION

Corresponding Author

Leticia Hosta-Rigau − Department of Health Technology, Centre for Nanomedicine and Theranostics, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark;

Email: [email protected]

Funding Sources

This work has been supported by the by the Danish Council for Independent Research [Grant No. 6111-00298B and 0130-00009B].

Conflict of Interest

The authors declare no conflict of interest

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Schematic 1 is available in the Supplementary Files section.

No competing interests reported.

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Schematic 1. Illustration of the preparation of haemoglobin (Hb) encapsulated metal-organic framework (MOF)-nanocarriers (NCs) endowed with antioxidant activity resulting from the incorporation of gold (Au)-based nanozymes.

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Metal-Organic Framework-Based Oxygen Carriers with Antioxidant Activity Resulting from the Incorporation of Gold-Based Nanozymes

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Hb Extraction from Bovine Blood

2.3. Fabrication and Characterization of Au-based NZs

2.4. Fabrication and Characterization of MOF-Nanocarriers (NCs) Loaded with Au-based NZs

2.5. Fabrication and Characterization of Hb-loaded Au@MOF-NCs (Au@MOF^Hb-NCs).

3. Results And Discussion

3.1. Fabrication and Characterization of Au-based NZs.

3.2. Catalytic Activity of the Au-based NZs

3.3. Fabrication and Characterization of Au@MOF-NCs

3.4. Catalytic Activity of the Au@MOF-NCs

3.5. Fabrication and Characterization of Au@MOF^Hb-NCs.

3.6. CAT-like activity of Au@MOF^Hb-NCs and Antioxidant Protection towards Conversion into metHb

4. Conclusions

Declarations

References

Schematic

Additional Declarations

Supplementary Files

Status:

Version 1

Metal-Organic Framework-Based Oxygen Carriers with Antioxidant Activity Resulting from the Incorporation of Gold-Based Nanozymes

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Hb Extraction from Bovine Blood

2.3. Fabrication and Characterization of Au-based NZs

2.4. Fabrication and Characterization of MOF-Nanocarriers (NCs) Loaded with Au-based NZs

2.5. Fabrication and Characterization of Hb-loaded Au@MOF-NCs (Au@MOFHb-NCs).

3. Results And Discussion

3.1. Fabrication and Characterization of Au-based NZs.

3.2. Catalytic Activity of the Au-based NZs

3.3. Fabrication and Characterization of Au@MOF-NCs

3.4. Catalytic Activity of the Au@MOF-NCs

3.5. Fabrication and Characterization of Au@MOFHb-NCs.

3.6. CAT-like activity of Au@MOFHb-NCs and Antioxidant Protection towards Conversion into metHb

4. Conclusions

Declarations

References

Schematic

Additional Declarations

Supplementary Files

Status:

Version 1

2.5. Fabrication and Characterization of Hb-loaded Au@MOF-NCs (Au@MOF^Hb-NCs).

3.5. Fabrication and Characterization of Au@MOF^Hb-NCs.

3.6. CAT-like activity of Au@MOF^Hb-NCs and Antioxidant Protection towards Conversion into metHb