A cancer cell membrane-encapsulated MnO2 nanoreactor for combined photodynamic-starvation therapy.

We demonstrate a MnO2-based nanoreactor to achieve continuous oxygen generation and efficient conversion from glucose to singlet oxygen for combined photodynamic-starvation therapy.

Glucose oxidase and polydopamine functionalized iron oxide nanoparticles: combination of the photothermal effect and reactive oxygen species generation for dual-modality selective cancer therapy.

Cancer cells possess some inherent characteristics, such as glucose-dependence and intolerance to heat and exogenous reactive oxygen species (ROS). In this study, a strategy has been developed to target these vulnerable weaknesses of cancer cells using glucose oxidase (GOx) and polydopamine (PDA) functionalized iron oxide nanoparticles (Fe3O4@PDA/GOx NPs). PDA is first deposited on the surfaces of iron oxide NPs through self-polymerization, and then GOx is covalently linked with PDA upon mixing the enzyme and Fe3O4@PDA under alkaline conditions. In this system, the PDA layer along with iron oxide NPs serves as a photothermal transfer material converting near infrared (NIR) radiation into heat. The covalently linked GOx can competitively consume glucose and spontaneously generate ROS H2O2 that can be further converted by the iron oxide NPs into more toxic ˙OH, inducing apoptosis of cancer cells. The selective toxicity of Fe3O4@PDA/GOx NPs on cancer cells is demonstrated both in vitro and in vivo. In particular, a single injection rather than multiple doses results in significant suppression of tumors, and does not induce apparent histological lesions in the 4T1 tumor-bearing Balb/c mice. The versatility of the functionalization strategy reported in this study will contribute to developing efficient therapies for selective cancer treatment.

Efficient fructose production from plant extracts by immobilized inulinases from Kluyveromyces marxianus and Helianthus tuberosus.

The enzymatic hydrolysis of poly- and oligosaccharides from plants seems like an advantageous approach for sugars production. Two inulinases producing fructose from plant oligosaccharides were isolated from yeast Kluyveromyces marxianus and plant Helianthus tuberosus. Both enzymes were immobilized on polymeric carriers by using the static adsorption approach. We could save 80.4% of the initial catalytic activity of plant inulinase immobilized on KU-2 cation-exchange resin and 75.5% of yeast enzyme activity adsorbed on AV-17-2P anion-exchange resin. After immobilization, the Km values increased 1.5 and 6 times for enzymes from K. marxianus and H. tuberosus, respectively. The optimal temperatures for catalysis of both enzymes were increased from 48-50 °C up to 70 °C. The activities of both immobilized enzymes remained unchanged after the 10 cycles of 20-min hydrolysis reaction at 70 °C model batch reactor. Sorbents, native and immobilized enzymes did not exhibit any mutagenic or cytotoxic activity.

Caco-2 Cell Conditions Enabling Studies of Drug Absorption from Digestible Lipid-Based Formulations.

PURPOSE: To identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs). METHODS: Caco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer. RESULTS: Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion. CONCLUSIONS: Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.

Immobilized lignin peroxidase from Ganoderma lucidum IBL-05 with improved dye decolorization and cytotoxicity reduction properties.

Use of free microbial enzymes for bioremediation and other industrial applications has several disadvantages like low stability and non-reusability in repeated batch operations. Immobilized enzymes are stable, recoverable and reusable in industrial processes. In this scenario G. lucidum IBL-05 LiP was entrapped in Ca-alginate beads using optimum concentrations of Na-alginate (4%), calcium chloride (0.2M) and glutraldehyde (0.02%). Optimum pH (pH 5) and temperature (55°C) for entrapped LiP were improved as compared to free LiP. Catalytic behavior of LiP also significantly enhanced, as Km value (0.25mM) decreased and Vmax value (868.6µmol/min) increased after ca-alginate entrapment of LiP. Decolorization efficiencies of Sandal reactive dyes after treating with immobilized LiP were in the range of 80-93%. A significant reduction was observed in water quality parameters including, BOD (66.44-98.22%), COD (81.34-98.82%) and TOC (80.21-97.77%) values. The cytotoxicity values for heamolytic and brine shrimp lethality of dye solutions treated with Ca-alginate immobilized LiP reduced up to 2.10-5.06% and 5.43-9.23%, respectively. On the basis of reduced toxicity and cytotoxicity values, it was concluded that Ca-alginate beads entrapped LiP may be an effective biocatalyst for bioremediation of dye based textile industry effluents.

Characteristic features and dye degrading capability of agar-agar gel immobilized manganese peroxidase.

Immobilization of enzymes has been regarded as an efficient approach to develop biocatalyst with improved activity and stability characteristics under reaction conditions. In the present study, purified manganese peroxidase (MnP) from Ganoderma lucidum IBL-05 was immobilized in agar-agar support using entrapment technique. Maximum immobilization yield was accomplished at 4.0% agar-agar gel. The immobilized MnP exhibited better resistance to changes in pH and temperature than the free enzyme, with optimal conditions being pH 6.0 and 50 °C. The kinetic parameters Km and Kcat/Km for free and entrapped MnP were calculated to be 65.6 mM and 6.99 M(-1) s(-1), and 82 mM and 8.15 M(-1) s(-1), respectively. Thermo-stability was significantly improved after immobilization. After 120 h, the insolubilized MnP retained its activity up to 71.9% and 60.3% at 30 °C and 40 °C, respectively. It showed activity until 10th cycle and retained 74.3% residual activity after 3th cycle. The effects of H2O2, ionic strength and potential inhibitors on activity of free and immobilized enzyme were investigated. Moreover, the decolorization of three structurally different dyes was monitored in order to assess the degrading capability of the entrapped MnP. The decolorization efficiencies for all the tested dyes were 78.6-84.7% after 12h. The studies concluded that the toxicity of dyes aqueous solutions was significantly reduced after treatment. The remarkable catalytic, thermo-stability and re-cycling features of the agar-agar immobilized MnP display a high potential for biotechnological applications.

Oral treatment for jaundice using immobilized bilirubin oxidase.

Jaundice is characterized by an excessive accumulation of bilirubin in the blood and tissues. A novel approach to reduce plasma levels of bilirubin by blocking its enterohepatic circulation was investigated. The treatment consisted of oral administration of immobilized bilirubin oxidase, which could oxidize bilirubin in the intestine to less toxic and more water-soluble products. In vivo administration of 0.1 to 2.0 mg/day of immobilized enzyme over a four-day period to chronically jaundiced Gunn rats effectively lowered plasma bilirubin levels, but only when the molar ratio of total serum bilirubin to rat serum albumin (B/RSA) was larger than 0.35. Plasma bilirubin concentration decreased in that group from an initial value of 11.3 to 6.3 mg/dl (-40%, n = 5) after eight days. This decrease was statistically significant (p < 0.05 by Student's t test). However, administration of bilirubin oxidase to rats with a B/RSA ratio less than 0.35 (n = 10) resulted in no statistically significant change in plasma bilirubin concentration.

Subchronic feeding study in rats of immobilized lactase following in utero exposure.

The effect of subchronic feeding of immobilized lactase composite following in utero exposure was studied in Sprague-Dawley rats. The dose levels of immobilized lactase composite that were used were 25, 100 and 400 mg per kg body weight per day. Reproduction performance, body weight gain and food consumption, organ weight, hematological and clinical chemical data, and ophthalmic, gross and histopathological examinations were used to study possible toxicological or pathological effects. No treatment-related effects were observed in either male or female rats exposed to immobilized lactase composite at any dose level.

In vivo safety of hollow fiber enzyme-reactors with immobilized phenylalanine ammonia-lyase in a large animal model for phenylketonuria.

Hollow fiber enzyme-reactors with immobilized phenylalanine ammonia-lyase (PAL) were developed for the in vivo depletion of phenylalanine (Phe) in circulating blood. A series of experiments was conducted with a large animal model in order to explore its safety for clinical use. The level of red blood cells, white blood cells and platelets did not change during a 2-hr application of the reactors in anesthetized, heparinized dogs and monkeys with experimental hyperphenylalaninemia. No increase in blood urea nitrogen was observed due to generation of ammonia from PAL-catalyzed Phe breakdown. The other metabolic product, trans-cinnamic acid, was reported to be nontoxic. Repeated application of the PAL-reactors to the same animals did not produce untoward physiological or immunological reactions. These data suggest that PAL-reactors may be safe for in vivo use to control excess Phe brought about by fever, infection or pregnancy in phenylketonuric individuals otherwise balanced by a Phe-poor diet. Application of PAL-reactors may serve as a model for extracorporeal enzyme replacement in enzyme-deficiency diseases.