Hybrid Protein Nano-Reactors Enable Simultaneous Increments of Tumor Oxygenation and Iodine-131 Delivery for Enhanced Radionuclide Therapy.

It is hard for current radionuclide therapy to render solid tumors desirable therapeutic efficacy owing to insufficient tumor-targeted delivery of radionuclides and severe tumor hypoxia. In this study, a biocompatible hybrid protein nanoreactor composed of human serum albumin (HSA) and catalase (CAT) molecules is constructed via glutaraldehyde-mediated crosslinking. The obtained HSA-CAT nanoreactors (NRs) show retained and well-protected enzyme stability in catalyzing the decomposition of H2 O2 and enable efficient labeling of therapeutic radionuclide iodine-131 (131 I). Then, it is uncovered that such HSA-CAT NRs after being intravenously injected into tumor-bearing mice exhibit efficient passive tumor accumulation as vividly visualized under the fluorescence imaging system and gamma camera. As the result, such HSA-CAT NRs upon tumor accumulation would significantly attenuate tumor hypoxia by decomposing endogenous H2 O2 produced by cancer cells to molecular oxygen, and thereby remarkably improve the therapeutic efficacy of radionuclide 131 I. This study highlights the concise preparation of biocompatible protein nanoreactors with efficient tumor homing and hypoxia attenuation capacities, thus enabling greatly improved tumor radionuclide therapy with promising potential for future clinical translation.

An Antioxidant Enzyme Therapeutic for COVID-19.

The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. Herein it is a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process, is reported. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.

Protein-drug conjugate programmed by pH-reversible linker for tumor hypoxia relief and enhanced cancer combination therapy.

Combining functional proteins with small molecular drugs into one entity may endow distinct synergistic advantages. However, on account of completely different physicochemical properties of such payloads, co-delivery through systemic administration for therapeutic purpose is challenging. Herein, we designed the protein-drug conjugate HSAP-DC-CAT (human serum albumin/Pt (IV)-dibenzocyclooctyne/chlorin e6-catalase) by modification of CAT and cisplatin pro-drug loaded HSA with pH-sensitive azide linker 3-(azidomethyl)-4-methyl-2,5-furandione (AzMMMan) followed by click chemistry assembly with DC. The dynamic covalent bonds between linker and proteins, on the one hand, can bridge proteins and small molecular drugs in the intermediate state for systemic delivery in the harsh in vivo environment; on the other hand, it can trigger traceless cleavage and release of drugs and proteins with full bioactivity in acidic microenvironment of tumor. The multifunctional HSAP-DC-CAT provides efficient cytosolic transduction in vitro, excellent blood half-lives after systemic administration, and significant antitumor outcome via integrated cisplatin-based chemotherapy and Ce6-based photodynamic therapy enhanced by catalase-induced manipulation of tumor hypoxia microenvironment. This study describes a universal formulation strategy for protein and small molecular drug by a bifunctional linker through amide reaction and click chemistry, with traceless in vivo release of therapeutic units.

Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties.

Polyhemoglobin-superoxide dismutase-catalase is designed to function as an oxygen carrier with antioxidant properties. This is based on cross-linking hemoglobin with superoxide dismutase and catalase (PolyHb-SOD-CAT). This study describes the structural and antioxidant properties of this solution. Our studies show that superoxide dismutase and catalase retain their enzymatic activity following glutaraldehyde polymerization with 8:1 and 16:1 glutaraldehyde:hemoglobin ratio. We have analyzed the optimal SOD/CAT ratios to prevent oxidation of hemoglobin in the presence of oxygen free radicals. The circulation half-life of crosslinked hemoglobin, SOD, and catalase in Sprague-Dawley rats correlates with the degree of polymerization as determined by high-performance molecular weight gel filtration. PolyHb-SOD-CAT decreases the formation of oxygen radicals compared with PolyHb in a rat intestinal ischemia-reperfusion model.

Novel catalase loaded nanocores for the treatment of inflammatory bowel diseases.

Inflammatory bowel disease (IBD) is an inflammatory disorder of the digestive tract reported to be primarily caused by oxidative stress. In this study, alginate encapsulated nanoceramic carriers were designed to deliver acid labile antioxidant enzyme catalase orally. Complete system was characterized for size, loading efficiency, in vitro antioxidant assay and in vitro release. The prepared nanoceramic system was found to be spherical with diameter of 925 ± 6.81 nm. The in vitro release data followed the Higuchi model in acidic buffer whereas in alkaline pH sustained and almost first order release of enzyme was observed up to 6 h.

Macrophages with cellular backpacks for targeted drug delivery to the brain.

Most potent therapeutics are unable to cross the blood-brain barrier following systemic administration, which necessitates the development of unconventional, clinically applicable drug delivery systems. With the given challenges, biologically active vehicles are crucial to accomplishing this task. We now report a new method for drug delivery that utilizes living cells as vehicles for drug carriage across the blood brain barrier. Cellular backpacks, 7-10 µm diameter polymer patches of a few hundred nanometers in thickness, are a potentially interesting approach, because they can act as drug depots that travel with the cell-carrier, without being phagocytized. Backpacks loaded with a potent antioxidant, catalase, were attached to autologous macrophages and systemically administered into mice with brain inflammation. Using inflammatory response cells enabled targeted drug transport to the inflamed brain. Furthermore, catalase-loaded backpacks demonstrated potent therapeutic effects deactivating free radicals released by activated microglia in vitro. This approach for drug carriage and release can accelerate the development of new drug formulations for all the neurodegenerative disorders.

Transglutaminase-catalyzed site-specific glycosidation of catalase with aminated dextran.

An enzymatic approach, based on a transglutaminase-catalyzed coupling reaction, was investigated to modify bovine liver catalase with an end-group aminated dextran derivative. We demonstrated that catalase activity increased after enzymatic glycosidation and that the conjugate was 3.8-fold more stable to thermal inactivation at 55 degrees C and 2-fold more resistant to proteolytic degradation by trypsin. Moreover, the transglutaminase-mediated modification also improved the pharmacokinetics behavior of catalase, increasing 2.5-fold its plasma half-life time and reducing 3-fold the total clearance after its i.v. administration in rats.

Inhibition of metastatic tumor growth by targeted delivery of antioxidant enzymes.

To develop effective anti-metastatic therapy, targeted or sustained delivery of catalase was examined in mice. We found that mouse lung with metastatic colonies of adenocarcinoma colon26 cells exhibited reduced catalase activity. The interaction of the tumor cells with macrophages or hepatocytes generated detectable amounts of ROS, and increased the activity of matrix metalloproteinases. Hepatocyte-targeted delivery of catalase was successfully achieved by galactosylation, which was highly effective in inhibiting the hepatic metastasis of colon26 cells. PEGylation, which increased the retention of catalase in the circulation, effectively inhibited the pulmonary metastasis of the cells. To examine which processes in tumor metastasis are inhibited by catalase derivatives, the tissue distribution and proliferation of tumor cells in mice was quantitatively analyzed using firefly luciferase-expressing tumor cells. An injection of PEG-catalase just before the inoculation of melanoma B16-BL6/Luc cells significantly reduced the number of the tumor cells in the lung at 24 h. Daily dosing of PEG-catalase greatly inhibited the proliferation of the tumor cells, and increased the survival rate of the tumor-bearing mice. These results indicate that targeted or sustained delivery of catalase to sites where tumor cells metastasize is a promising approach for inhibiting metastatic tumor growth.

Effect of ethanol in vivo on enzymes which detoxify oxygen free radicals.

The effects of ethanol administered as a 15% solution in drinking fluid on weight gain, soluble liver protein and the activity of the three enzymes of oxygen radical metabolism (i.e., superoxide dismutase, catalase, and glutathione peroxidase) were studied in five inbred strains of mice (129/ReJ, BALB/c, C3H/HeSnJ, C57BL/6J, Csb) and Sprague Dawley rats, relative to age, sex, and genotype matched controls. Animals maintained on ethanol exhibited lower weight gains and elevation of soluble liver protein than controls. Total superoxide dismutase, catalase and glutathione peroxidase activity in ethanol-treated animals were in general reduced in comparison to that of their matched controls, with each strain showing genotype specific enzyme activity. Such ethanol feeding results are attributed to the direct and indirect effects of this treatment protocol and raise the possibility that ethanol-fed animals may be susceptible to free radical damage and at least some of the cellular damages observed following ethanol challenges could be attributed to the reduced level of these protective enzymes.

Modulated insulin permeation across a glucose-sensitive polymeric composite membrane.

A glucose-sensitive polymeric composite membrane was prepared based on our previously developed stimuli-responsive membrane system. Membranes were cast from a mixture of glucose oxidase (GOD), catalase, and poly(N-isopropylacrylamide-co-methacrylic acid) (poly(NIPAm/MAA)) nanoparticles dispersed in a solution of a hydrophobic polymer. High efficiency of enzyme immobilization was achieved with undetectable leakage. The bioactivity of the immobilized GOD, as measured by pH change of glucose solutions, was found to be equivalent to approximately 80% of that of the free GOD. The addition of catalase markedly increased the oxidation rate of glucose. However, an optimal unit ratio of GOD to catalase and optimal enzyme loading were observed. The rate of insulin permeation through the membrane was modulated by glucose concentration due to shrinking or swelling of the embedded pH-sensitive nanoparticles. The response of insulin permeability to the change in the glucose concentration could be detected within 5-15 min. The permeability of insulin increased more than 3-fold as the glucose concentration was raised from 50 to 200 mg/dl. The average insulin permeability at 400 mg/dl of glucose was 8-fold that at 50 mg/dl in a continuous test in saline and was 6-fold in a three-cycle discontinuous test in pH 7.4 buffer.

Ionic adsorption of catalase on bioskin: kinetic and ultrastructural studies.

Bioskin is a natural polymer produced by Acetobacter xylinum and several yeasts in culture. It contains glucosamine and N-acetyl galactosamine which promote ionic adsorption of catalase at the adequate pH value. High values of ionic strength are required to enzyme desorption. Adsorption of catalase on bioskin fibers has been visualized by scanning electron microscopy associated to a dispersion X-ray analyzer. At low enzyme density, the affinity of the immobilized catalase for hydrogen peroxide was 30% lower than that of the free enzyme. This affinity decreased dramatically at higher density of immobilized enzyme and could not be increased by agitation of the enzyme reaction mixture. Immobilized catalase retains about 70% of its initial activity after 16 d storage, whereas soluble enzyme is completely inactivated after 3 d at room temperature. The haeme group of catalase is not protected after immobilization since it is accessible to both EDTA and phloroglucinol, chelating agents which inactivate catalase by removing the iron atom from the haeme group.

Hepatocyte-specific distribution of catalase and its inhibitory effect on hepatic ischemia/reperfusion injury in mice.

To explore the possibility of using catalase for the treatment of reactive oxygen species (ROS)-mediated injuries, the pharmacokinetics of bovine liver catalase (CAT) labeled with 111In was investigated in mice. At a dose of 0.1 mg/kg, more than 70% of 111In-CAT was recovered in the liver within 10 min after intravenous injection. In addition, 111In-CAT was predominantly recovered from the parenchymal cells (PC) in the liver. Increasing the dose retarded the hepatic uptake of 111In-CAT, suggesting saturation of the uptake process. This cell-specific uptake could not be inhibited by coadministration of various compounds which are known to be taken up by liver PC, indicating that the uptake mechanism of CAT by PC is very specific to this compound. The preventive effect of CAT on a hepatic ischemia/reperfusion injury was examined in mice by measuring the GOT and GPT levels in plasma. A bolus injection of CAT at 5 min prior to the reperfusion attenuated the increase in the levels of these indicators in a dose-dependent manner. These results suggest that catalase can be used for various hepatic injuries caused by ROS.

Morphologic changes induced by short-term ischemia in the rat testis are not affected by treatment with superoxide dismutase and catalase.

Short-term testicular ischemia was induced unilaterally by ligating the testicular artery for 60 or 100 minutes in adult rats. After 7 days, the rats were fixed by vascular perfusion. The effect of ischemia and reperfusion was quantified using morphometric techniques. Occlusion of the testicular artery for 60 and 100 minutes resulted in a mild and moderate damage, respectively, to the seminiferous tubules. The contralateral control testis was not affected. To study the role of oxygen radicals in ischemia-reperfusion-induced testicular damage, rats were treated with superoxide dismutase and catalase. These radical scavengers did not influence the extent of testicular damage.

Catalase-associated abnormalities and H2O2 increase in pre-neoplastic and neoplastic lesions of the human lower female genital tract and their near adjacent epithelia.

We report that an internal and non-UV-dependent type of neoplasia, the human cervical intraepithelial neoplasia (SIL), is also deficient in catalase activity, like the UV-induced tumors in the autosomal recessive human epithelial disease, xeroderma pigmentosum (XP). Whether or not the lesions are papillomavirus (HPV) positive in the different categories of preneoplastic and neoplastic extracts, the following parameters are affected: i), catalase activity level; ii), kinetic profile of catalase activity; iii), H2O2 increase. Mathematical treatment of these parameters (CONSTEL-Program), unambiguously distinguishes between normal and pathological cases. Such analyses make it possible to grade the pathological samples into 4 classes, depending on their deviance from normality. These classes may be correlated with the gradual steps in the process of malignant transformation defined by histological and clinical diagnosis. We found conformity between catalase activity and histological analyses in 66 biopsies, out of a total of 100 biopsies (35 patients). Moreover, 23 patients presenting decreased catalase activities in 31 biopsies showed disease progression after 3 to 6 months contrary to surgery histological data. We show that ATP synthesis in the presence of catalase and H2O2 (further aspect of catalase function), may occur in neoplastic extracts at much lower concentrations of H2O2 than in normal extracts. Thus, the catalase abnormality seems to be a good tool to study pre-neoplastic to neoplastic evolution of lesions and their adjacent tissues of the lower female genital tract; furthermore, i) it provides an earlier, more powerful means of detecting micro-SIL in progression to squamous cell carcinoma, than combined clinical and histological examinations; ii) model for investigating drugs such as in situ H2O2 scavengers or agents increasing glutathione peroxidase activity (GSH).

Effect of allopurinol or superoxide dismutase plus catalase on cardiovascular function after burn injury in the anaesthetized rat.

A scald burn injury was applied to 22-23 per cent of the body surface area of anaesthetized rats. The cardiac output was 38-60 per cent lower in the burn injury group than in the control group; heart rate and mean arterial blood pressure were only slightly affected, but burn injury caused a significant increase in total peripheral resistance. The involvement of oxygen-free radicals in this immediate fall in cardiac output was investigated. Pretreatment with a blocker of free radical production, allopurinol or the infusion of the free radical scavengers superoxide dismutase plus catalase caused no cardiovascular improvement, suggesting that oxygen free radicals are not involved in the fall in cardiac output after burn injury. Allopurinol treatment, however, prevented the rise in total peripheral resistance seen after burn injury.

Biodegradable microspheres as carriers for native superoxide dismutase and catalase delivery.

The purpose of this research was to encapsulate superoxide dismutase (SOD) and catalase (CAT) in biodegradable microspheres (MS) to obtain suitable sustained protein delivery. A modified water/oil/water double emulsion method was used for poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) PLA MS preparation co-encapsulating mannitol, trehalose, and PEG400 for protein stabilization. Size, morphology, porosity, mass loss, mass balance, in vitro release and in vitro activity were assessed by using BCA protein assay, scanning electron microscopy, BET surface area, and particle-sizing techniques. In vitro activity retention within MS was evaluated by nicotinammide adenine dinucleotide oxidation and H2O2 consumption assays. SOD encapsulation efficiency resulted in 30% to 34% for PLA MS and up to 51% for PLGA MS, whereas CAT encapsulation was 34% and 45% for PLGA and PLA MS, respectively. All MS were spherical with a smooth surface and low porosity. Particle mean diameters ranged from 10 to 17 mum. CAT release was prolonged, but the results were incomplete for both PLA and PLGA MS, whereas SOD was completely released from PLGA MS in a sustained manner after 2 months. CAT results were less stable and showed a stronger interaction than SOD with the polymers. Mass loss and mass balance correlated well with the release profiles. SOD and CAT in vitro activity was preserved in all the preparations, and SOD was better stabilized in PLGA MS. PLGA MS can be useful for SOD delivery in its native form and is promising as a new depot system.

Development of bone-targeted catalase derivatives for inhibition of bone metastasis of tumor cells in mice.

Removal of hydrogen peroxide by delivering catalase to the vicinity of metastasizing tumor cells is a promising approach for inhibiting tumor metastasis. To inhibit bone metastasis, catalase was conjugated with 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (Bip), a derivative of bone-seeking bisphosphonates, polyethylene glycol (PEG), or both. Bip-conjugated catalase derivatives, that is, catalase-Bip and PEG-catalase-Bip, exhibited a higher affinity for bone matrix as compared with their counterparts without Bip. The tissue distribution of (111) In-labeled catalase derivatives indicated that the accumulation of radioactivity in bones was increased by conjugation of either Bip or PEG with catalase. An experimental bone metastasis model was developed by injecting male C57BL/6 mice with murine melanoma B16-BL6/Luc cells, which stably express firefly luciferase into left ventricle. Repeated injections of catalase to tumor-bearing mice had no significant effect on the number of melanoma cells in tibiae and femurs, whereas injections of catalase-Bip, PEG-catalase, or PEG-catalase-Bip significantly reduced the number. These results indicate that targeted delivery of catalase to the bones can be achieved by conjugating the enzyme with either Bip or PEG, and this delivery is effective in inhibiting the bone metastasis of tumor cells.

Blood-borne macrophage-neural cell interactions hitchhike on endosome networks for cell-based nanozyme brain delivery.

BACKGROUND: Macrophage-carried nanoformulated catalase ('nanozyme') attenuates neuroinflammation and protects nigrostriatal neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. This is facilitated by effective enzyme transfer from blood-borne macrophages to adjacent endothelial cells and neurons leading to the decomposition of reactive oxygen species. MATERIALS & METHODS: We examined the intra- and inter-cellular trafficking mechanisms of nanozymes by confocal microscopy. Improved neuronal survival mediated by nanozyme-loaded macrophages was demonstrated by fluorescence-activated cell sorting. RESULTS: In macrophages, nanozymes were internalized mainly by clathrin-mediated endocytosis then trafficked to recycling endosomes. The enzyme is subsequently released in exosomes facilitated by bridging conduits. Nanozyme transfer from macrophages to adjacent cells by endocytosis-independent mechanisms diffusing broadly throughout the recipient cells. In contrast, macrophage-free nanozymes were localized in lysosomes following endocytic entry. CONCLUSION: Facilitated transfer of nanozyme from cell to cell can improve neuroprotection against oxidative stress commonly seen during neurodegenerative disease processes.

Kinetic study on the effects of extremely low frequency electromagnetic field on catalase, cytochrome P450 and inducible nitric oxide synthase in human HaCaT and THP-1 cell lines.

Extremely low frequency electromagnetic fields (ELF-EMF) have been found to produce a variety of biological effects. These effects of ELF-EMF depend upon frequency, amplitude, and length of exposure, and are also related to intrinsic susceptibility and responsiveness of different cell types. Although the mechanism of this interaction is still obscure, ELF-EMF can influence cell proliferation, differentiation, cell cycle, apoptosis, DNA replication and protein expression. The aim of this study was to estimate various kinetic constants of catalase, cytochrome P450 and inducible nitric oxide synthase in response to ELF-EMF exposure in human HaCaT and THP-1 cell lines. In order to evaluate the effect of ELF-EMF on the modulation of cellular responses to an inflammatory stimulus, both cell lines were treated with lipopolysaccharide. To the best of our knowledge there is no available report on such type of kinetic study of selected enzymes in response to ELF-EMF in these cell lines. Therefore, the current study may reveal novel mechanism of ELFEMF biological interaction with the enzymological and hormonal systems of living organisms. These new insights may be important for ELF-EMF application particularly for wound healing, tissue regeneration, Parkinson's and Alzheimer's diseases.

Macrophage delivery of therapeutic nanozymes in a murine model of Parkinson's disease.

BACKGROUND: Parkinson's disease is a common progressive neurodegenerative disorder associated with profound nigrostriatal degeneration. Regrettably, no therapies are currently available that can attenuate disease progression. To this end, we developed a cell-based nanoformulation delivery system using the antioxidant enzyme catalase to attenuate neuroinflammatory processes linked to neuronal death. METHODS: Nanoformulated catalase was obtained by coupling catalase to a synthetic polyelectrolyte of opposite charge, leading to the formation of a polyion complex micelle. The nanozyme was loaded into bone marrow macrophages and its transport to the substantia nigra pars compacta was evaluated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. RESULTS: Therapeutic efficacy of bone marrow macrophages loaded with nanozyme was confirmed by twofold reductions in microgliosis as measured by CD11b expression. A twofold increase in tyrosine hydroxylase-expressing dopaminergic neurons was detected in nanozyme-treated compared with untreated MPTP-intoxicated mice. Neuronal survival was confirmed by magnetic resonance spectroscopic imaging. Bone marrow macrophage-loaded catalase showed sustained release of the enzyme in plasma. CONCLUSION: These data support the importance of macrophage-based nanozyme carriage for Parkinson's disease therapies.