Transport of Zinc-Phthalocyanine to Cancer Cells using Myoglobin-Albumin Fusion Protein for Photodynamic Therapy.

Photodynamic therapy (PDT) is a noninvasive approach to cancer treatment, wherein cell death is initiated by singlet oxygen (1O2) production via energy transfer from excited photosensitizers to ground-state O2. Effective clinical photosensitizers necessitate water solubility for in vivo administration. Hydrophobic dyes, such as phthalocyanines, cannot be used directly as photosensitizers. Herein, we synthesized a myoglobin-(human serum albumin) fusion protein reconstituted with zinc-phthalocyanine (ZnPc), termed ZnPcMb-HSA. The photophysical properties of ZnPcMb-HSA closely resemble those of ZnPc-substituted Mb. Notably, ZnPc dissociates from ZnPcMb-HSA and selectively accumulates within cancer cells, while the protein components remain extracellular. Treatment of four distinct cell lines with ZnPcMb-HSA, followed by red-light irradiation, effectively induced apoptosis. The half-maximal inhibitory concentrations (IC50) against these cancer cell lines ranged between 0.1-0.5 µM. Reconstituted Mb-HSA emerges as a promising carrier for transporting various water-insoluble porphyrinoid photosensitizer to target cancer cells in PDT applications.

Methemoglobin-albumin clusters for cyanide detoxification.

Sodium nitrite (NaNO2) is a universal antidote for patients with cyanide poisoning. However, its use has serious drawbacks in terms of efficacy and safety. Herein, we present a promising antidote: methemoglobin (metHb)-albumin clusters. The metHb-albumin cluster is made by a metHb core wrapped by covalently bound human serum albumin. Spectral analyses proved that the metHb-albumin clusters possessed cyanide-binding properties similar to those of naked metHb. In vitro cell experiments showed that metHb-albumin clusters prevented the cyanide-induced inhibition of cytochrome c oxidase activity, resulting in a strong cytoprotective effect. In mice subjected to cyanide poisoning, metHb-albumin clusters reduced mortality and alleviated metabolic acidosis, while maintaining the activity of cytochrome c oxidase in organs; their efficacy was better than that of NaNO2. Furthermore, the oxygen carrying capacity was maintained in poisoned mice treated with metHb-albumin clusters and was low in those treated with NaNO2. These results indicate that metHb-albumin clusters could be a more effective and safer antidote against cyanide poisoning than NaNO2.

Zinc Substituted Myoglobin-Albumin Fusion Protein: A Photosensitizer for Cancer Therapy.

Myoglobin combined with human serum albumin (Mb-HSA) can be produced using yeast Pichia pastoris as a host strain, with secretion into the culture medium. This Mb-HSA fusion protein possesses identical O2 binding affinity to that of naked Mb. The Mb unit is reconstituted with a zinc(II) protoporphyrin IX, yielding (zinc substituted Mb)-HSA, ZnMb-HSA. The photophysical property and singlet O2 generation ability of ZnMb-HSA are equivalent to those of ZnMb. In vitro cell experiments revealed that ZnMb-HSA acts as a superior photosensitizer for photodynamic cancer therapy. It is noteworthy that ZnMb-HSA shows long circulation lifetime in vivo.

Methemoglobin-Albumin Cluster Incorporating Protoporphyrin IX: Dual Functional Protein Drug for Photodynamic Therapy.

We describe the synthesis, photophysical properties, and photodynamic activity of a methemoglobin (metHb) wrapped covalently by human serum albumins (HSAs) incorporating protoporphyrin IX (PPIX): a metHb-HSA3 -PPIX2 cluster. The metHb core catalyzes H2 O2 disproportionation to generate O2 in tumor tissue. The HSA3 -PPIX2 shell acts as a photosensitizer for 1 O2 formation. The metHb-HSA3 -PPIX2 cluster acts as a dual functional protein drug for photodynamic therapy.

Rectangular Holes in Porphyrin Isomers Act As Mono- and Binucleating Ligands: Stereochemistry of Mono- and Diboron Porphycenes and Their Protonation Behaviors.

The first boron complexes of porphycenes, structural isomers of porphyrin, are reported. They are synthesized in good yields by reacting the free-base porphycene ligands with BF3·Et2O through a microwave-assisted method. Depending on the substituent group of porphycenes, two different coordination structures, mono- and diboron porphycenes, are obtained simultaneously. The single crystal structures and DFT calculations suggest that the boron atom of the monoboron porphycene is favorably coordinated on the dipyrroethene site, and the regioisomer of diboron porphycene is of cisoid stereochemistry, which is more stable than transoid. We also investigate the protonation behavior of boron porphycene complexes. Diboron porphycene does not undergo protonation, whereas monoboron porphycene undergoes protonation at the nonboron coordinating pyrroline site, resulting in a red shift in both absorption and emission spectra. Protonation and deprotonation of monoboron porphycene can be reversibly triggered using acids and bases.

Hemoglobin(ßC93A)-Albumin Cluster: Mutation of Cysteine-ß93 to Alanine Allows Moderate Reduction of O2 Affinity by Inositol Hexaphosphate.

Covalent wrapping of recombinant human hemoglobin (Cys-ß93→Ala) variant rHb(ßC93A) by human serum albumin (HSA) yielded the rHb(ßC93A)-HSA3 cluster as an artificial O2 carrier as a red blood cell substitute. Complexation of inositol hexaphosphate to the central rHb(ßC93A) core reduced the O2 affinity moderately, in much the same way as that of naked hemoglobin. This reduction might be attributable to the inert, small Ala-ß93 residue, which cannot be reacted with the bulky maleimide crosslinker.

Novel Hemoglobin-Based Oxygen Carrier Bound With Albumin Shows Neuroprotection With Possible Antioxidant Effects.

Background and Purpose- A hemoglobin-albumin cluster, 1 core of hemoglobin covalently bound with 3 shell albumins, designated as HemoAct was developed as a hemoglobin-based oxygen carrier. We aim to investigate neuroprotection by HemoAct in transient cerebral ischemia and elucidate its underlying mechanisms. Methods- Male rats were subjected to 2-hour transient middle cerebral artery occlusion and were then administered HemoAct transarterially at the onset of reperfusion. Neurological and pathological findings were examined after 24 hours of reperfusion to identify neuroprotection by HemoAct. Intermittent measurements of cortical blood flow and oxygen content were performed, and a histopathologic analysis was conducted on rats during the early phase of reperfusion to assess the therapeutic mechanism of HemoAct. In addition, the antioxidant effects of HemoAct were examined in hypoxia/reoxygenation-treated rat brain microvascular endothelial cells. Results- Neurological deterioration, infarct and edema development, and the activation of MMP-9 (matrix metalloprotease-9) and lipid peroxidation after 24 hours of reperfusion were significantly ameliorated by the HemoAct treatment. Reductions in blood flow and tissue partial oxygen pressure in the cortical penumbra after 6 hours of reperfusion were significantly ameliorated by the HemoAct treatment. The histopathologic analysis of the cortical penumbra revealed that HemoAct in HemoAct-treated rats showed superior microvascular perfusion with the mitigation of microvascular narrowing changes than autologous erythrocytes in nontreated rats. Although HemoAct extravasated into the ischemic core with serum protein, it did not induce an increase in serum extravasation or reactive oxygen species production in the ischemic core. In vitro experiments with rat brain microvascular endothelial cells revealed that HemoAct significantly suppressed cellular reactive oxygen species production in hypoxia/reoxygenation-treated cells, similar to albumin. Conclusions- HemoAct exerted robust neuroprotection in transient cerebral ischemia. Superior microvascular perfusion with an oxygen delivery capability and possible antioxidant effects appear to be the underlying neuroprotective mechanisms.

Self-Propelled Soft Protein Microtubes with a Pt Nanoparticle Interior Surface.

Human serum albumin (HSA) microtubes with an interior surface composed of Pt nanoparticles (PtNPs) are self-propelled in aqueous H2 O2 medium. They can capture cyanine dye and Escherichia coli (E. coli) efficiently. Microtubes were prepared by wet templating synthesis by using a track-etched polycarbonate (PC) membrane with alternate filtrations of aqueous HSA, poly-l-arginine (PLA), and citrate-PtNPs. Subsequent dissolution of the PC template yielded uniform hollow cylinders made of (PLA/HSA)8 PLA/PtNP stacking layers (1.16±0.02 µm outer diameter, ca. 23 µm length). In aqueous H2 O2 media, the soft protein microtubes are self-propelled by jetting O2 bubbles from the open-end terminus. The effects of H2 O2 and surfactant concentrations on the velocity were investigated. The swimming microtube captured cyanine dye in the HSA component of the wall. Addition of an intermediate γ-Fe3 O4 layer allowed manipulation of the direction of movement of the tubule by using a magnetic field. Because the exterior surface is positively charged, the bubble-propelled microtubes adsorbed E. coli with high efficiency. The removal ratio of E. coli by a single treatment reached 99 %.

Zinc-Substituted Hemoglobin-Albumin Cluster as a Porphyrin-Carrier for Enhanced Photodynamic Therapy.

Apohemoprotein is focused on the field of theranostics, serving as a porphyrin carrier. Hemoglobin (Hb) consists of α2ß2 tetramer with iron(II)-protoporphyrin IX (heme) bound to each globin. However, heme-removed Hb (apoHb) causes dissociation at αß interfaces and aggregation under physiological conditions. We synthesized a stable apoHb derivative comprising intramolecular-crosslinked apoHb (apoXHb) and human serum albumin (HSA), apoXHb-HSA3. ApoXHb-HSA3 engendered no aggregates in the physiological solutions. Moreover, apoXHb-HSA3 was reconstituted with zinc(II)-protoporphyrin IX (ZnP), generating ZnXHb-HSA3, a potent photosensitizer for photodynamic therapy (PDT). The photophysical properties of ZnXHb-HSA3 were identical to those of zinc-substituted XHb (ZnXHb). Cellular uptake behavior was evaluated using various cancer cell lines. ZnXHb-HSA3 released ZnP around the cells, and the free ZnP penetrated cell membranes. In contrast, protein units were not observed within the cells. ZnXHb-HSA3 showed no cytotoxicity under dark conditions and demonstrated superior PDT activity in comparison to naked ZnXHb. ZnXHb-HSA3 acts as an innovative porphyrin carrier for enhanced PDT.

Dual delivery of carbon monoxide and doxorubicin using haemoglobin-albumin cluster: proof of concept for well-tolerated cancer therapy.

A serious concern of doxorubicin (DOX) therapy is that it causes severe adverse effects, particularly cardiotoxicity. Carbon monoxide (CO) possesses powerful cytoprotective effects against drug-induced organ injury and is expected to ameliorate DOX-induced cardiotoxicity. In this study, a dual carrier of DOX and CO (CO-HemoAct-DOX) was fabricated based on a haemoglobin-albumin cluster (HemoAct), which is a protein cluster with a haemoglobin core structure wrapped by serum albumin. CO-HemoAct-DOX was synthesised by binding CO to a haemoglobin core and covalently conjugating (6-maleimidocaproyl)hydrazone derivative of DOX to an albumin shell. The average DOX/cluster ratio was about 2.6. In the in vitro cytotoxicity assay against cancer cells, the anti-tumour activity of CO-HemoAct-DOX was 10-fold lower than that of DOX in a 2D-cultured model, whereas CO-HemoAct-DOX suppressed the growth of tumour spheroids to the same extent as DOX in the 3D-cultured model. In colon-26 tumour-bearing mice, CO-HemoAct-DOX achieved DOX delivery to the tumour site and alleviated tumour growth more effectively than DOX. Furthermore, CO-HemoAct attenuated DOX-induced cardiomyocyte atrophy in H9c2 cells and elevated the levels of cardiac biomarkers in mice exposed to DOX. These results suggest that the dual delivery of CO and DOX using HemoAct is a promising strategy as an anti-tumour agent to realise well-tolerated cancer therapy with minimal cardiotoxicity.

Gold nanoparticle inclusion into protein nanotube as a layered wall component.

We describe the synthesis, structure, and catalytic activity of human serum albumin (HSA) nanotubes (NTs) including gold nanoparticles (AuNPs) as a layered wall component. The NTs were fabricated as an alternating layer-by-layer assembly of AuNP and HSA admixture (a negatively charged part) and poly-l-arginine (PLA, a positively charged part) into a track-etched polycarbonate membrane (400 nm pore diameter) with subsequent dissolution of the template. SEM images showed the formation of uniform hollow cylinders of (PLA/AuNP-HSA)3 with a 426 ± 12 nm outer diameter and 65 ± 7 nm wall thickness. Transmission electron microscopy and energy dispersive X-ray measurements revealed high loading of AuNPs in the tubular wall. HSAs bind strongly onto the individual AuNP (K = 1.25 × 10(9) M(-1)), generating a core-shell AuNP-HSA corona, which is the requirement of the robust NT formation. Calcination of the (PLA/AuNP-HSA)3 NTs at 500 °C under air yielded red solid NTs composed of thermally fused AuNPs. From the mass decrease by heat treatment, we calculated the weight of the organic components (PLA and HSA) and thereby constructed a six-layer model of the tube. The (PLA/AuNP-HSA)3 NTs serve as a heterogeneous catalyst for reduction of 4-nitrophenol with sodium borohydrate. Furthermore, implantation of the stiff (PLA/AuNP-HSA)3 NTs vertically onto glass plate produced uniformly cylindrical tube arrays.

Covalent core-shell architecture of hemoglobin and human serum albumin as an artificial O2 carrier.

Covalent core-shell structured protein clusters of hemoglobin (Hb) and human serum albumin (HSA) (HbX-HSAm) (m = 2, 3) with novel physiological properties were generated by linkage of Hb surface lysins to HSA cysteine-34 via an α-succinimidyl-ε-maleimide cross-linker (X: 1 or 2). The isoelectric points of HbX-HSAm (pI = 5.0-5.2) were markedly lower than that of Hb and almost identical to that of HSA. AFM and TEM measurements revealed a triangular Hb1-HSA3 cluster in aqueous medium. The complete 3D structure of Hb1-HSA3 based on TEM data was reconstructed, revealing two possible conformer variants. All HbX-HSAm clusters showed a moderately higher O2 affinity than the native Hb. Furthermore, the exterior HSA units possess a remarkable ability to bind lumiflavin (LF). The addition of NADH to an aqueous solution of the met-Hb2-(HSA-LF)3 cluster reduced the inactive ferric Hb center to the functional ferrous Hb. This O2-carrying hemoprotein cluster with strongly negative surface net charge, high O2 affinity, and NADH-dependent reductase unit can support a new generation of molecular architecture for red blood cell substitutes.

Protein-Porphyrin Complex Photosensitizers for Anticancer and Antimicrobial Photodynamic Therapies.

Photodynamic therapy (PDT) efficiently induces apoptosis through visible-light irradiation of photosensitizers (PSs) within tumors and microbial cells. Porphyrin analogues serve as widely utilized photosensitizing agents with their therapeutic abilities being governed by molecular structures and central metal ions. However, these macrocyclic compounds tend to agglutinate and form stacks in aqueous environments, resulting in a loss of photochemical activity. To overcome this limitation, encapsulation within liposomes and polymer micelles enables the dispersion of porphyrins as monomolecular entities in aqueous solutions, preventing undesirable deactivation. Recently, the use of reconstituted hemoproteins containing various metal-porphyrins and protein cages incorporating porphyrins has garnered significant interest as a new generation of biocompatible PSs. In this concept paper, we provide a comprehensive review of recent developments and trends of protein-porphyrin complex PSs for applications in anticancer and antimicrobial PDTs.

Pharmaceutical Integrity of Lyophilized Methemoglobin-Albumin Clusters after Reconstitution.

Covalent attachment of a ferric hemoglobin (metHb) core to three human serum albumin molecules to form metHb-albumin clusters has previously been used to develop an antidote for hydrogen sulfide poisoning. Lyophilization is one of the most effective approaches to preserve protein pharmaceuticals with minimum contamination and decomposition. However, there is concern that lyophilized proteins may undergo pharmaceutical alteration on reconstitution. This study investigated the pharmaceutical integrity of metHb-albumin clusters on lyophilization and reconstitution with three clinically available reconstitution fluids, (i) sterile water for injection, (ii) 0.9% sodium chloride injection, and (iii) 5% dextrose injection. The metHb-albumin clusters retained their physicochemical properties and structural integrity on lyophilization and reconstitution with sterile water for injection or 0.9% sodium chloride injection, along with comparable hydrogen sulfide scavenging ability compared to non-lyophilized metHb-albumin clusters. The reconstituted protein completely rescued lethal hydrogen sulfide poisoning in mice. On the other hand, lyophilized metHb-albumin clusters reconstituted with 5% dextrose injection showed physicochemical changes and a higher mortality rate in mice subjected to lethal hydrogen sulfide poisoning. In conclusion, lyophilization represents a potent preservation method for metHb-albumin clusters if either sterile water for injection or 0.9% sodium chloride injection is used for reconstitution.

Pharmaceutical stability of methemoglobin-albumin cluster as an antidote for hydrogen sulfide poisoning after one-year storage in freeze-dried form.

Long-term stability during storage is an important requirement for pharmaceutical preparations. The methemoglobin (metHb)-albumin cluster, in which bovine metHb is covalently enveloped with an average of three human albumin molecules, is a promising antidote for hydrogen sulfide (H2S) poisoning. In this study, we investigated the pharmaceutical stability of metHb-albumin cluster after storage for one year in solution and as freeze-dried powder. The lyophilized powder of metHb-albumin cluster stored for one year was readily reconstituted in sterile water for injection, yielding a homogeneous brown solution. Physicochemical measurements revealed that the overall structure of the metHb-albumin cluster was still maintained after preservation. Results of the pharmacological study showed that 100 % of the H2S-poisoned mice survived after treatment with the reconstituted solution of metHb-albumin cluster powder. Furthermore, the solution did not cause any toxic reactions. The antidotal efficacy of metHb-albumin cluster for H2S poisoning was preserved in freeze-dried powder form for at least one year.

Polyoxazoline-Conjugated l-Asparaginase: An Antibody-Production-Free Therapeutic Agent for Acute Lymphoblastic Leukemia.

l-asparaginase (ASNase), an enzyme that catalyzes the hydrolysis of l-asparagine into l-aspartic acid, is frequently used as a medication for acute lymphoblastic leukemia (ALL). However, when derived from bacterial sources, this enzyme can elicit side effects, including allergic or hypersensitivity reactions, owing to immune responses. Here, we describe the synthesis of polyoxazoline-conjugated ASNase (POx-ASNase) and investigate its enzyme activity, anticancer efficacy, immunogenicity, and retention in the bloodstream. The water-soluble POx was coupled with surface lysine residues of ASNase using a bifunctional cross-linker. The average number of polymers bound to each enzyme was determined as 10. Although the enzymatic activity of POx-ASNase decreased to 56% of that of native ASNase, its temperature and pH dependencies remained unaltered. Remarkably, the lyophilized powder form of POx-ASNase retained its catalytic ability for 24 months. POx-ASNase demonstrated nearly identical anticancer efficacy compared to naked ASNase against leukemia and lymphoma cells (MOLT-4, CLBL-1, and K562) while displaying no cytotoxicity toward normal cells. Animal experiments conducted using rats revealed that the POx decoration suppressed the generation of anti-ASNase IgM and IgG antibodies with no detection of anti-POx antibodies. The half-life within the bloodstream extended to 34 h, representing a 17-fold increase compared to unmodified ASNase. These findings suggest that POx-ASNase serves as an anticancer therapeutic agent, characterized by the absence of antibody production and notably extended circulation persistence.

Poly(2-ethyl-2-oxazoline)-Conjugated Hemoglobins as a Red Blood Cell Substitute.

Hemoglobin wrapped covalently with poly(2-ethyl-2-oxazoline)s (POx-Hb) is characterized physicochemically and physiologically as an artificial O2 carrier for use as a red blood cell (RBC) substitute. The POx-Hb is generated by linkage of porcine Hb surface-lysines to a sulfhydryl terminus of the POx derivative, with the average binding number of the polymers ascertained as 6. The POx-Hb shows moderately higher colloid osmotic activity and O2 affinity than the naked Hb. Human adult HbA conjugated with POx also possesses equivalent features and O2 binding properties. The POx-Hb solution exhibits good hemocompatibility, with no influence on the functions of platelets, granulocytes, and monocytes. Its circulation half-life in rats is 14 times longer than that of naked Hb. Hemorrhagic shock in rats is relieved sufficiently by infusion of the POx-Hb solution, as revealed by improvements of circulatory parameters. Serum biochemistry tests and histopathological observations indicate no acute toxicity or abnormality in the related organs. All results indicate that POx-Hb represents an attractive alternative for RBCs and a useful O2 therapeutic reagent in transfusion medicine.

Neuroprotective effects of a hemoglobin-based oxygen carrier (stroma-free hemoglobin nanoparticle) on ischemia reperfusion injury.

The application of hemoglobin (Hb)-based oxygen carriers (HBOCs) to the treatment of cerebral ischemia has been investigated. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA3) was found to exert neuroprotective effects on ischemia/reperfusion injury. Stroma-free hemoglobin nanoparticles (SFHbNP), a subsequently developed HBOC consisting of a spherical polymerized stroma-free Hb core with a HSA shell, contains the natural antioxidant enzyme catalase and, thus, is expected to exert additive effects. We herein investigated whether SFHbNP exerted enhanced neuroprotective effects in a rat transient middle cerebral artery occlusion (tMCAO) model. Rats were subjected to 2-hour tMCAO and divided into the following 3 groups with the intravenous administration of the respective reagents: (1) phosphate-buffered saline (PBS), as a vehicle (2) Hb-HSA3, and (3) SFHbNP. After 24-hour reperfusion, infarct and edema volumes decreased in the order of the PBS, Hb-HSA3, and SFHbNP groups, with a significant difference (p < 0.05) between the PBS and SFHbNP groups. Similar reductions were observed in oxidative stress, leukocyte recruitment, and blood-brain barrier disruption in the order of the PBS, Hb-HSA3, and SFHbNP groups. In the early phase of reperfusion within 6 h, microvascular HBOC perfusion and cerebral blood flow were maintained at high levels during the reperfusion period in the Hb-HSA3 and SFHbNP groups. However, a difference was observed in tissue oxygen partial pressure levels, which significantly decreased after 6-hour reperfusion in the Hb-HSA3 group, but remained high in the SFHbNP group. A superior oxygen transport ability appears to be related to the enhanced neuroprotective effects of SFHbNP.

Polyoxazoline-conjugated porcine serum albumin as an artificial plasma expander for dogs.

Veterinary medicine has made tremendous progress for domestic dogs, which are irreplaceable family members enriching human life. Nevertheless, no adequate supply system exists for their blood products. This study examined the synthesis, structure, safety, and efficacy of poly(2-ethyl-2-oxazoline)-conjugated porcine serum albumin (POx-PSA) as an artificial plasma expander for dogs. The aqueous POx-PSA solution showed moderately high colloid osmotic pressure and good blood cell compatibility. Actually, lyophilized powder stored for 1 year can regenerate into a homogeneous solution. The circulation half-life of POx-PSA in rats was 2.1-fold longer than that of naked PSA. Rats produced neither anti-PSA IgG antibody nor anti-POx IgG antibody, which suggests excellent immunological stealth properties of POx-PSA. Complete resuscitation of hemorrhagic shock in rats was achieved soon after injection of POx-PSA solution. Serum biochemistry tests and histopathological observations indicated no abnormality in the related organs. When POx-PSA was administered to dogs intravenously, (i) no serum biochemical or hematological alteration was observed, also (ii) no overt deterioration of animal health was observed. These results indicate that POx-PSA has potential as an artificial plasma expander for dogs.

Methemoglobin-albumin clusters for the treatment of hydrogen sulfide intoxication.

Hydrogen sulfide (H2S) has attracted significant attention as a seed in drug development. However, H2S is toxic and induces lethal acute intoxication. Here, we developed methemoglobin (metHb)-albumin clusters as detoxifying agents for H2S intoxication, which were designed based on the inherent binding property of metHb with H2S. The metHb-albumin clusters comprising an autoxidized ferric Hb center wrapped covalently with an average of three human serum albumins showed a similar H2S binding affinity to that of naked metHb. Owing to the H2S binding capability, metHb-albumin clusters suppressed cell death induced by H2S exposure while maintaining mitochondrial function in H9c2 cells. In addition, lethal H2S intoxication model mice were rescued by a single administration of metHb-albumin clusters, resulting from the recovery of cytochrome c oxidase activity. Furthermore, the metHb-albumin clusters possessed essential characteristics, such as adequate pharmacokinetic properties and biocompatibility, for their use as detoxifying agents against H2S intoxication. In conclusion, the results obtained in this study suggest that metHb-albumin clusters are promising detoxifying agents for H2S intoxication and that harnessing the inherent H2S binding properties of metHb is an innovative approach to develop detoxifying agents for H2S intoxication.