Bacterial nanocellulose loaded with bromelain and nisin as a promising bioactive material for wound debridement.

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles.

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.

Triggering Sequential Catalytic Fenton Reaction on 2D MXenes for Hyperthermia-Augmented Synergistic Nanocatalytic Cancer Therapy.

The unique characteristics of a tumor microenvironment (TME) enable the development of new tumor-therapeutic modalities with high efficiency, biosafety, and tumor specificity. In this work, we report on the construction of photothermal-enhanced and nanocatalyst-enabled sequential catalytic reaction for TME-specific cancer therapy. This conceptual advance is achieved by engineering the surface of two-dimensional Ti3C2 MXene with two separate catalysts, including natural glucose oxidase (GOD) as glucose catalysts and superparamagnetic iron oxide nanoparticles (IONPs) as Fenton-reaction nanocatalysts. A sequential catalytic reaction is triggered by using GOD for catalyzing the tumor-overtaken glucose to generate large amounts of hydrogen peroxide molecules. Subsequently IONPs can catalyze the transformation of pregenerated hydrogen peroxide into large amounts of highly toxic hydroxyl radicals to kill the cancer cells subsequently in TME-enabled acidity condition. The two-dimensional (2D) Ti3C2 MXene matrix efficiently converts the near-infrared light into thermal energy to synergistically enhance the catalytic efficiency of this sequential catalytic reaction and therefore achieve the high synergistic cancer-therapeutic outcome, accompanied with the high biocompatibility of the constructed composite nanocatalysts. Both in vitro cancer-cell evaluation and in vivo tumor xenograft on nude mice with complete tumor eradication demonstrate the high synergistic efficiency of photothermal-enhanced sequential nanocatalytic cancer therapy. Therefore, this work substantially broadens the biomedical applications of 2D MXenes to nanocatalytic cancer therapy by enhancing the Fenton reaction-based nanocatalytic therapy via converting the near-infrared light into thermal energy and subsequently elevating the local Fenton-reaction temperature.

Anti-inflammatory and antifibrotic effects of a combination of spiperone and immobilized hyaluronidase on partially reversible and irreversible toxic pneumofibrosis.

The possibility of boosting antifibrotic activity of testicular hyaluronidase immobilized on polyethylene oxide with spiperone was studied on the bleomycin models of a single (partially reversible pneumofibrosis) and repeated (irreversible pneumofibrosis) injuries to the alveolar epithelium in C57Bl/6 mice. The antifibrotic effect was more pronounced after successive treatment with immobilized hyaluronidase and spiperone than after individual treatment with each of the compounds: no collagen deposition in the parenchyma of bleomycin-damaged lungs was found. The decrease in inflammatory cell (lymphocytes, macrophages, neutrophils, plasma cells) infiltration of the alveoli and alveolar tracts interstitium in mice treated by immobilized hyaluronidase and spiperone did not differ from the anti-inflammatory effect of spiperone monotherapy.

Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment.

A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA-pectin cryogels and enzyme release was 20.8±2.1%, 63.8±0.2%, 41.5±3.5% and 26.0±3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37°C after 3h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5±1.5, 65.8±3.8, 57.3±0.2 and 34.0±4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA-pectin cryogels at pH close to the human skin (pH=5.5) showed 15.0% free antibiotic following first order kinetic at 37°C after 5h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.

Evaluation of whole lysosomal enzymes directly immobilized on titanium (IV) oxide used in the development of antimicrobial agents.

Lysosomal enzymes isolated from egg white were directly immobilized on titanium (IV) oxide (TiO(2)) particles using shaking methods (150 rpm, room temperature, 10 min), and the immobilization efficiency, activity, and stability of lysosomal enzymes immobilized on TiO(2) were evaluated. Of the various mass ratios (w/w) of lysosomal enzymes to TiO(2) tested, we found that 100% immobilization efficiency was observed at a ratio of 1:20 (enzymes:TiO(2); w/w). Furthermore, the antimicrobial activities of the immobilized lysosomal enzymes were confirmed using viable cell counts against Escherichia coli. Our results showed that the antimicrobial activity of immobilized lysosomal enzymes is stable and can be maintained up to one month, but the antimicrobial activity of free enzymes without immobilization completely disappeared after five days in storage. In addition, enhanced immobilization efficiency was shown in TiO(2) pretreated with a divalent, positively charged ion, Ca(2+), and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized enzymes. However, K(+), a monovalent, positively charged ion, did not have any positive effect on immobilization or antimicrobial activity. Finally, we suggest that activity and stability of immobilized lysosomal enzymes can be maintained for a longer time than those properties of free lysosomal enzymes.

Alkaline phosphatase-polyresorcinol complex: characterization and application to seed coating.

An alkaline phosphatase (EC 3.1.3.1) from Escherichia coli ATCC27257 was immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retained about 74% of the original enzymatic activity. The pH and temperature profile of the immobilized and free enzyme revealed a similar behavior. Kinetic parameters were determined: K(m) and K(i) values were 2.44 and 0.423 mM, respectively, for the phosphatase-polyresorcinol complex and 1.07 and 0.069 mM, respectively, for free phosphatase. The thermal and storage stabilities of the immobilized phosphatase were higher than those of the native one. On addition to soil, free enzyme was completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex was comparatively stable. Barley seed coated with the immobilized enzyme exhibited higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus was detected when the seed was encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot was observed.

Biological evaluation of calcium alginate microspheres as a vehicle for the localized delivery of a therapeutic enzyme.

Gaucher disease (GD) is caused by the decreased activity and/or stability of the lysosomal enzyme glucocerebrosidase (GCR). The available treatment consists in the intravenous administration of exogenous GCR, and is effective in reverting most of the symptoms. However, in terms of bone pathology, which is among the most disabling manifestations, a slow and incomplete response is observed, indicating that adjuvant therapies are necessary to consistently restore GCR activity in bone and accelerate regeneration. In this study, calcium alginate microspheres were analyzed as a vehicle for localized GCR delivery to bone. Results demonstrated that the entrapped enzyme retained full activity and exhibited a broader pH-dependent activity profile, compared to that of free-GCR, with improved stability at physiological pH. GCR release profile was established, and it was demonstrated that GCR could be released in a sustained manner. The biological behavior of the system was evaluated by analyzing the uptake of released GCR by GCR-deficient cells from GD patients, using different techniques: GCR activity measurements, radiolabeling, and cellulose acetate electrophoresis. Results demonstrated that GCR was internalized by cells significantly enhancing the residual enzymatic activity. To achieve an activity reconstitution level comparable to that obtained using free-GCR, only half of the dose was required with entrapped-GCR.

[Conditions for immobilizing protosubtilin on aluminum oxide using transitional metal ions]. / Issledovanie uslovii immobilizatsii protosubtilina na okisi aliuminiia pri pomoshchi ionov perekhodnykh metallov.

The effect of the degree of the carrier activation by titanium tetrachloride, type of buffer solution, and protein concentration on characteristics of protosubtiline G10x immobilized on alumina was studied. X-ray phase analysis and IR spectroscopy were used to investigate the structure of the carrier and its changes during activation. To obtain the immobilized enzyme preparation with the maximum activity, immobilization should be carried out in 0.02 M Ca-acetate buffer on alumina treated with 2% solution of titanium tetrachloride.

[Immobilized luciferase of Luciola mingrelica fireflies. The kinetic properties and thermostability of luciferase immobilized on cellulose films]. / Immobilizovannaia liutsiferaza svetliakov Luciola mingrelica. Kineticheskie svoistva i termostabil'nost' liutsiferazy, immobilizovannoi na tselliuloznykh plenkakh.

Luciferase of fireflies Luciola mingrelica was immobilized on cellulose films activated by cyanuric chloride or sodium periodate. Kinetic properties and the contribution of diffusional obstacles to the kinetics of the immobilized enzyme were examined. External and internal diffusion were found to influence the kinetic parameters. The stability of the enzyme was investigated at 25 degrees C and pH 7.8. Thermoactivation of the immobilized enzyme was shown to proceed in two stages: fast and slow. Dithiotreitol and cystein stabilized the enzyme at the fast stage while salt supplements at both stages. The fast thermoinactivation stage was apparently associated with the oxidation of luciferase SH-groups. It is demonstrated that the immobilized enzyme of Luciola mingrelica can be employed to measure ATP traces with the detection limit 0.1 mM. The enzyme immobilized on cellulose films can be used repeatedly.