Complementary autophagy inhibition and glucose metabolism with rattle-structured polydopamine@mesoporous silica nanoparticles for augmented low-temperature photothermal therapy and in vivo photoacoustic imaging.

Rattle-structured nanoparticles with movable cores, porous shells and hollow interiors have shown great effectiveness in drug delivery and cancer theranostics. Targeting autophagy and glucose have provided alternative strategies for cancer intervention therapy. Herein, rattle-structured polydopamine@mesoporous silica nanoparticles were prepared for in vivo photoacoustic (PA) imaging and augmented low-temperature photothermal therapy (PTT) via complementary autophagy inhibition and glucose metabolism. Methods: The multifunctional rattle-structured nanoparticles were designed with the nanocore of PDA and the nanoshell of hollow mesoporous silica (PDA@hm) via a four-step process. PDA@hm was then loaded with autophagy inhibitor chloroquine (CQ) and conjugated with glucose consumer glucose oxidase (GOx) (PDA@hm@CQ@GOx), forming a corona-like structure nanoparticle. Results: The CQ and GOx were loaded into the cavity and decorated onto the surface of PDA@hm, respectively. The GOx-mediated tumor starvation strategy would directly suppress the expression of HSP70 and HSP90, resulting in an enhanced low-temperature PTT induced by PDA nanocore. In addition, autophagy inhibition by the released CQ made up for the loss of low-temperature PTT and starvation efficiencies by PTT- and starvation-activated autophagy, realizing augmented therapy efficacy. Furthermore, the PDA nanocore in the PDA@hm@CQ@GOx could be also used for PA imaging. Conclusion: Such a "drugs" loaded rattle-structured nanoparticle could be used for augmented low-temperature PTT through complementarily regulating glucose metabolism and inhibiting autophagy and in vivo photoacoustic imaging.

Novel polymeric microspheres: Synthesis, enzyme immobilization, antimutagenic activity, and antimicrobial evaluation against pathogenic microorganisms.

New polymeric microspheres containing azomethine (1a-1c and 2a-2c) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT-IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis-Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (Vmax ) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well-diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.

Oncolytic Nanoreactors Producing Hydrogen Peroxide for Oxidative Cancer Therapy.

In situ generation of anticancer agents at the place of the disease is a new paradigm for cancer therapy. The production of highly potent drugs by nanoreactors through a facile synthesis pathway is demanded. We report an oncolytic nanoreactor platform loaded with the enzyme glucose oxidase (GOX) to produce hydrogen peroxide. For the first time, we realized a core-shell structure with encapsulated GOX under mild synthetic conditions, which ensured high remaining activity of GOX inside of the nanoreactor. Moreover, the nanoreactor protected the loaded GOX from proteolysis and contributed to increased thermal stability of the enzyme. The nanoreactors were effectively taken up into different cancer cells, in which they produced hydrogen peroxide by consuming intracellular glucose and oxygen, thereby leading to effective death of the cancer cells. In summary, our robust nanoreactors are a promising platform for effective anticancer therapy and sustained enzyme utilization.

Optimal initial state for fast parameter estimation in nonlinear dynamical systems.

BACKGROUND AND OBJECTIVE: This paper deals with the improvement of parameter estimation in terms of precision and computational time for dynamical models in a bounded error context. METHODS: To improve parameter estimation, an optimal initial state design is proposed combined with a contractor. This contractor is based on a volumetric criterion and an original condition initializing this contractor is given. Based on a sensitivity analysis, our optimal initial state design methodology consists in searching the minimum value of a proposed criterion for the interested parameters. In our framework, the uncertainty (on measurement noise and parameters) is supposed unknown but belongs to known bounded intervals. Thus guaranteed state and sensitivity estimation have been considered. An elementary effect analysis on the number of sampling times is also implemented to achieve the fast and guaranteed parameter estimation. RESULTS: The whole procedure is applied to a pharmacokinetics model and simulation results are given. CONCLUSIONS: The good improvement of parameter estimation in terms of computational time and precision for the case study highlights the potential of the proposed methodology.

Characterization of a microsphere formulation containing glucose oxidase and its in vivo efficacy in a murine solid tumor model.

PURPOSE: This work focused on the characterization and in vitro/in vivo evaluation of an alginate/chitosan microsphere (ACMS) formulation of glucose oxidase (GOX) for the locoregional delivery of reactive oxygen species for the treatment of solid tumors. METHODS: The GOX distribution and ACMS composition were determined by confocal laser scanning microscopy and X-ray photoelectron spectroscopy. The mechanism of GOX loading and GOX-polymer interactions were examined with Fourier transform infrared spectroscopy and differential scanning calorimetry. In vitro cytotoxicity and in vivo efficacy of GOX-encapsulated ACMS (ACMS-GOX) were evaluated in EMT6 breast cancer cells and solid tumors. RESULTS: GOX was loaded into calcium alginate (CaAlg) gel beads via electrostatic interaction and the CaAlg-GOX-chitosan complexation likely stabilized GOX. Higher concentrations of GOX near the surface of ACMS were detected. GOX retained its integrity upon adsorption to CaAlg gel beads during the coating and after release from ACMS. ACMS-GOX exhibited cytotoxicity to the breast cancer cells in vitro and their efficacy increased with increasing incubation time. Intratumorally delivered ACMS-GOX significantly delayed tumor growth with much lower general toxicity than free GOX. CONCLUSION: The results suggest that the ACMS-GOX formulation has the potential for the intratumoral delivery of therapeutic proteins to treat solid tumors.

Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution.

Adsorption condition and enzymatic activity of glucose oxidase (GOD) on polystyrene (PS) film surfaces modified with ozone aeration and UV irradiation (O3/UV) treatment were investigated. The total amount of GOD immobilized on the PS film modified with the O3/UV treatment in distilled water (PS-W film) was approximately twice as large as that on the film treated in an aqueous ammonia solution (PS-A film), whereas the specific activity of GOD on the PS-A film was four times higher than that on the PS-W film. In contrast, no enzymatic activity of GOD on the non-treated PS film was observed because of irreversible denaturation of the adsorbed GOD. We therefore conclude that the PS films modified by the O3/UV treatment in the aqueous media are effective in immobilizing GOD.

Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity.

Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods: physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion-conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, zeta-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings: (1) the emulsion-conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion-conjugation technique (about 3.5 x 10(-)(5) AU microg(-)(1) s(-)(1)) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion-conjugation technique. As a result, the comparison of these three techniques showed the emulsion-conjugation technique to be a potentially effective and practical way to fabricate alginate/GOx microspheres for implantable glucose biosensor application.

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.

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells.

Cell-selective intracellular targeting is a key element of more specific and safe enzyme, toxin, and gene therapies. Endothelium poorly internalizes certain candidate carriers for vascular immunotargeting, such as antibodies to platelet endothelial cell adhesion molecule 1 (PECAM-1). Conjugation of poorly internalizable antibodies with streptavidin (SA) facilitates the intracellular uptake. Although both small and large (100-nm versus 1000-nm diameter) anti-PECAM/SA-beta galactosidase (SA-beta-gal) conjugates bound selectively to PECAM-expressing cells, only small conjugates showed intracellular accumulation of active beta-gal. To study whether size of the conjugates controls the uptake, a series of anti-PECAM/SA and anti-PECAM/bead conjugates ranging from 80 nm to 5 microm in diameter were produced. Human umbilical vein endothelial cells and PECAM-transfected mesothelioma cells internalized 80- to 350-nm anti-PECAM conjugates, but not conjugates larger than 500 nm. Further, size controls intracellular targeting of active therapeutic cargoes in vitro and in vivo. Small anti-PECAM/DNA conjugates transfected target cells in culture 5-fold more effectively than their large counterpart (350- versus 4200-nm diameter). To evaluate the practical significance of the size-controlled subcellular addressing, we coupled glucose oxidase (GOX) to anti-PECAM and antithrombomodulin. Both types of conjugates had equally high pulmonary uptake after intravenous injection in mice, yet only small (200- to 250-nm), not large (600- to 700-nm), GOX conjugates caused profound oxidative vascular injury in the lungs, presumably owing to intracellular generation of H(2)O(2). Thus, engineering of affinity carriers of specific size permits intracellular delivery of active cargoes to endothelium in vitro and in vivo, a paradigm useful for the targeting of drugs, genes, and toxins.

Immunoassay for native enzyme quantification in biological samples.

In order to detect low levels of enzyme activity, specifically glucose oxidase, in biological samples, an immunoenzymatic assay was developed since currently available methods could not be used because of either their lack of sensitivity or the conditions prevailing in our samples: turbidity of the medium, presence of redox systems other than glucose oxidase, and high concentration of proteins. The principle of the method is to coat a polystyrene surface with a fragment Fc-specific anti-IgG, then with an antibody directed against the looked-for enzyme, which is simultaneously the antigen and the enzyme activity required for immunoenzymatic detection. We applied this concept to biological samples after glucose oxidase administration to mice. This method achieves specificity and sensitivity (20 ng/mL or 1 ng) with samples of biological origin. No marker is needed since the antigen itself possesses an enzyme activity. This method, which requires a small sample volume (50 microL, 20 microL, if necessary), can be extended easily to the many enzymes currently used as markers. It could also be applied to the native enzymes of medical interest for which antibodies and a colorimetric reaction are available.

Effects of sonicated eosinophils on the in vitro sensitivity of human lymphoma cells to glucose oxidase.

We report here that cultured human lymphoma cells in the absence of sonicated eosinophils are sensitive to killing by glucose oxidase (beta-D-glucose:oxygen-oxido reductase; EC 1.1.3.4) at concentrations as low as 0.025 microgram/ml, a level that can be rapidly attained in s.c. tumor implants in mice that receive a single nonlethal injection of enzyme. Multiple clonogenic assays were used to measure the survival of human lymphoma cell lines (H9 and ARH-77) cultured for 14 days in complete RPMI 1640 supplemented with exogenous glucose oxidase (0.025-2.5 micrograms/ml) or an immunoconjugate containing glucose oxidase (0.25-25 micrograms/ml) in the presence or absence of catalase (10 micrograms/ml) or an equal number of sonicated human eosinophils with or without supplemental 100 microM Br-, I-, or SCN-. In addition, we used an immunoassay to measure the concentration of glucose oxidase in s.c. implants of the Sp 2/0 myeloma tumor at 0-30 min after an i.v. injection of 50 micrograms of enzyme into 21 BALB/c mice. Doses of glucose oxidase as small as 0.025 microgram/ml killed more than 3 logs of tumor cells. Catalase completely inhibited, and sonicated human eosinophils partially inhibited, the killing by glucose oxidase or immunoconjugate, whereas supplemental halides had no effect. Glucose oxidase i.v. produced levels > 0.04 microgram/g of tumor for 30 min after injection with a peak concentration of 0.079 microgram/g of tumor within 5 min of injection. These results are important because certain human lymphomas contain extensive extracellular deposits of eosinophil peroxidase, thereby making these tumors potentially less susceptible to killing by otherwise therapeutic doses of glucose oxidase.

Preclinical safety studies of glucose oxidase.

The purpose of this study was to develop preclinical safety data regarding the toxicologic and pharmacokinetic properties of glucose oxidase. Groups of adult BALB/c mice were injected with four doses of the enzyme ranging from 0.125 U of glucose oxidase/g b.wt. to 1 U/g b.wt., and the following responses were measured: survival, methemoglobin, glucose, blood urea nitrogen, creatine phosphokinase, erythrocyte count and body weight. We also compared the biodistribution of the enzyme in mice to the biodistribution of glucose oxidase conjugated to a monoclonal antibody. Finally, we assessed the histopathologic changes produced in mice by glucose oxidase and the binding of the enzyme to snap-frozen, human autopsy tissues. As expected, the acute toxicity of glucose oxidase was primarily due to methemoglobinemia (mean concentration 36% at the highest dose) and transient hypoglycemia (as low as 35 mg/dl). Furthermore, conjugated and unconjugated glucose oxidase had a blood half-life of less than 2 hr and concentrated in the liver and spleen. On the basis of our studies, we conclude that glucose oxidase has reasonably predictable toxicities and is, therefore, safe for human trials. The rapid uptake of conjugated and unconjugated glucose oxidase by the liver and spleen, however, may significantly limit the therapeutic targeting of glucose oxidase.

A new enzymatic assay for evaluating the clearance of immune complexes from the circulation of mice.

A new photometric in vivo enzymatic immune complex clearance (EIC) assay was developed in a homologous system using glucose oxidase-anti-glucose oxidase complexes (GAG) as a model of immune complexes. Chromatographically purified GAG was injected into mouse tail veins and at intervals thereafter the enzyme activities of GAG remaining in the circulation were estimated. The GAG were cleared in a size dependent manner and were stable, being eluted as the same discrete peaks on HPLC size-exclusion chromatography both before and after injection into mice. The complement consuming activity of the GAG was weak, and depletion of complement components with cobra-venom factor did not alter clearance of the GAG from the circulation, whereas pretreatment of aggregated mouse gamma globulin suppressed the clearance rate. These results suggested that most of the GAG were not cleared via complement receptors but via FcR. Normal clearance rates were significantly changed by administration of immunomodulators such as carrageenan or LPS. Intravenous administration of GAG at a dose 50 times higher than normal caused no deaths suggesting that the complexes were of low toxicity. The enzymatic method presented should be of value for measuring the function of the mononuclear phagocytic system with respect to immune complex clearance. It provides a rapid and sensitive alternative assay which avoids using radioisotopes.

In vivo administration of glucose oxidase conjugated with monoclonal antibodies to angiotensin-converting enzyme. The tissue distribution, blood clearance, and targeting into rat lungs.

A conjugate between glucose oxidase (GO) and monoclonal antibody to human angiotensin-converting enzyme (ACE) cross-reacting with rat ACE (MoAb9b9) has been prepared by oxidation of the cardohydrate moiety of the enzyme with sodium periodate. The conjugate (GO-MoAb9b9) thus obtained retained both antigen-binding capacity and enzymatic activity. The fate of the conjugate in vivo after intravenous injection was studied using conjugates containing radiolabeled enzyme. GO-MoAb9b9 was specifically accumulated in rat lungs upon in vivo administration, as compared with free enzyme and nonimmune IgG-conjugated glucose oxidase. The specificity of the conjugate accumulation expressed as the localization ratio (the ratio between radioactivity of gram tissue to that of blood) (Loc. Ratio) reached a value up to 50 on the second day after injection, in contrast to native enzyme and to IgG-conjugated enzyme (Loc. Ratio was less than 0.5 for both preparations). The Loc. Ratio of GO-MoAb9b9 was even higher than that of the original antibody MoAb9b9 and was equal to 20, which is probably explained by an extremely rapid blood clearance of the conjugate from the circulation. The administration of excess free MoAb9b9 dramatically inhibited the conjugate targeting in the lung without any effect on liver uptake. At doses ranging from 10 to 1,000 micrograms/rat, the conjugate was accumulated in the lung without saturation of the antigen determinants of the target. At minimal doses, the efficiency of targeting achieved 5 to 7% of the conjugate injected. With elevation of the dose, the efficiency of targeting decreased to 2.5% of the injected dose (1 mg of GO-MoAb9b9 per rat). A sixfold greater accumulation of unmodified radiolabeled MoAb9b9 compared with the GO-MoAb9b9 conjugate in rat lung has been observed, though the kinetics of desorption from the target organ was similar for both the antibody and the conjugate. In the bloodstream, the conjugate persisted for at least 5 days without binding to blood cells; all circulating radioactivity was associated with proteins. A considerable part of the conjugate (to 50%) circulated as a tight antibody-enzyme complex for several days. The conjugate retained its antigen-binding capacity for at least 24 h; during this period, its enzymatic activity decreased by less than 40%. The results obtained provide the experimental ground for further attempts to apply glucose oxidase conjugates for local modulation of inflammation and elimination of the target cells.

Effect of prosthetic sugar groups on the pharmacokinetics of glucose-oxidase.

The administration of enzymes is of potential therapeutic value in many disease states, e.g. lysosomal storage diseases, provided problems in the metabolism and targeting of large proteins can be overcome. We have addressed ourselves to these problems by studying the pharmacokinetics and distribution of glucose-oxidase (GO) and some of its derivatives in mice. A saturable mechanism was responsible for GO uptake by mononuclear phagocytes. After construction of a pharmacokinetic model, the Kuptake (850 nmol/l) and the number of capturing cells were determined; uptake was half the initial plasma concentration in about 10 min. Deglycosylated GO's had half-lives of about 100 min and were taken up by the same organs that took up native GO. Galactosylated GO had a half-life of 4 min and a different distribution; it was taken up preferentially by the liver in hepatocytes. Our results illustrate the role sugars might play in the targeting of foreign proteins to different cell types, and the feasibility of determining in vivo microscopic constants such as the affinity between molecules and certain cells.