Screening of Venlafaxine Hydrochloride for Transdermal Delivery: Passive Diffusion and Iontophoresis

The objective of the study was to investigate in vitro transdermal delivery of venlafaxine hydrochloride across the pigskin by passive diffusion and iontophoresis. For passive diffusion, experiments were carried out in Franz diffusion cell whereas for iontophoretic permeation, the diffusion cell was modified to contain both the donor and return electrode on the same side of skin. Anodal iontophoresis was carried out using a current density of 0.5 mA/cm². Donor concentrations used were 585.5 mg/ml (saturated solution) and 100 mg/ml. Experiments initially performed to determine the transport efficiency of venlafaxine ions showed promising results. Iontophoresis increased the permeation rate at both concentration levels over their passive counterparts (P < 0.01), but surprisingly higher steady-state flux was obtained from lower donor drug load (P < 0.01). The favorable pH of the unsaturated solutions is suggested to be the cause for this effect. Mild synergistic effect was observed when iontophoresis was carried out incorporating peppermint oil in the donor but the same was not found in passive diffusion. Highest steady-state flux obtained in the experiment was 3.279 μmol/cm²/h when peppermint oil (0.1%) was included in the donor. As the maintenance requirement of venlafaxine hydrochloride is approximately 9.956 μmol/h, the results suggested that the drug is a promising candidate for iontophoretic delivery.     

Phosphorylated chitosan membranes for the separation of ethanol-water mixtures by pervaporation

Dense membranes of chitosan were prepared and ionically crosslinked with phosphoric acid for varying intervals of time. The membranes were characterized by FTIR and XRD to confirm cross-linking. TGA and IEC studies were conducted to assess the thermal stability and estimate the number of interactive groups left in the membrane after crosslinking. Sorption studies were carried out to evaluate the extent of interaction and degree of swelling of the membranes in pure liquids as well as binary mixtures. The phosphorylated chitosan membrane crosslinked for 2 h showed good mechanical strength and strong potential for breaking the azeotrope of 95.58 wt% ethanol by exhibiting a high pervaporation selectivity of 213 with substantial water flux of 0.58 kg/(m² h). Pervaporation experimental parameters such as feed composition, membrane thickness and permeate pressure were varied to identify optimum operating conditions.     

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank’s solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb⁺ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb⁺ in the absence and in the presence of ouabain. The optimal conditions for Rb⁺ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 °C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC₅₀ was 100 μM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.     

Quantitative analysis of the bacteriophage Qβ infection cycle

In this study, the infection cycle of bacteriophage Qβ was investigated. Adsorption of bacteriophage Qβ to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4 × 10^− 10 ml/cells/min. In infected cells, approximately 130 molecules of β-subunit and 2 × 10⁵ molecules of coat protein were translated in 15 min. Replication of Qβ RNA proceeded in 2 steps—an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5 × 10⁵ molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.     

Preparation and characterization of chitosan-polyvinyl alcohol blend hydrogels for the controlled release of nano-insulin

Chitosan (CS)-polyvinyl alcohol (PVA) blend hydrogels were prepared using glutaraldehyde as the cross-linking agent. The obtained hydrogels, which have the advantages of both PVA and CS, can be used as a material for the transdermal drug delivery (TDD) of insulin. The nano-insulin-loaded hydrogels were prepared under the following conditions: 1.2 g of polyethylene glycol, 1.5 g of CS, 1.2 g of PVA, 1.2 mL of 1% glutaraldehyde solution, 16 mL of water, and 40 mg of nano-insulin with 12 min of mixing time and 3 min of cross-linking time. The nano-insulin-loaded hydrogels were characterized using scanning electron microscopy, energy dispersive spectrometry, Fourier-transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and its mechanical properties were analyzed. The results show that all molecules in the hydrogel have good compatibility and they formed a honeycomb-like structure. The hydrogel also showed good mechanical and thermal properties. The in vitro drug release of the hydrogel showed that the nano-insulin accorded with Fick's first law of diffusion and it has a high permeation rate (4.421 μg/(cm² h)). These results suggest that the nano-insulin-loaded hydrogels are a promising non-invasive TDD system for diabetes chemotherapy.     

Effects of taurine and hypotaurine supplementation and ionophore concentrations on post-thaw acrosome reaction of dog spermatozoa

The aim of this work was to study of the effect of the amino acids (AA) taurine (T) and hypotaurine (H) and of different calcium ionophore concentrations on the ability of capacitated frozen-thawed dog sperm to undergo the acrosome reaction (AR). Fifteen ejaculates grouped into five pools were used. Sperm was frozen at a concentration of 80 × 10⁶ sperm cells/mL in the Uppsala Equex extender (UE) supplemented with 25, 50 and 75 mM of either AA. The UE extender without T or H was used as control. After thawing, sperm was capacitated with Canine Capacitation Medium for 20 min. Sperm was then challenged with calcium ionophore A23178 at 0, 2.5 and 10 μM concentration and evaluated for integrity of plasma and acrosome membranes after 5, 15 and 30 min of incubation, utilizing PI/Fitc-PNA fluorescent staining and flow cytometry.Sperm cryopreserved in UE supplemented with 50 mM T (UE 50T) had higher AR rates than sperm cryopreserved with UE 75T, UE 25H and UE 50H, but AR rates were similar to semen frozen with the control extender. Challenges with 2.5 and 10 μM/L of calcium ionophore increased AR in frozen-thawed sperm incubated for 5, 15 and 30 min. The combination of calcium ionophore concentration and incubation time resulting in the highest AR rate was 10 μM and 15 min.     

Lateral pressure dependence of the phospholipid transmembrane diffusion rate in planar-supported lipid bilayers

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10⁻⁴ s⁻¹ to 1.03 × 10⁻⁴ s⁻¹) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å²/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.     

The rate of spontaneous cleavage of the glycosidic bond of adenosine

Previous estimates of the rate of spontaneous cleavage of the glycosidic bond of adenosine were determined by extrapolating the rates of the acid- and base-catalyzed reactions to neutral pH. Here we show that cleavage also proceeds through a pH-independent mechanism. Rate constants were determined as a function of temperature at pH 7 and a linear Arrhenius plot was constructed. Uncatalyzed cleavage occurs with a rate constant of 3.7 × 10⁻¹² s⁻¹ at 25 °C, and the rate enhancement generated by the corresponding glycoside hydrolase is ∼5 × 10¹²-fold.     

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant K_A = 7.0(± 0.8) × 10⁴ M^− 1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.     

Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-l-maurocalcine analog

L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to ¹²⁵I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9 × 10⁻⁷ μl. ¹²⁵I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of ¹²⁵I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5 min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24 h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Separation and purification of phycocyanin from Spirulina sp. using a membrane process

The highest purity ratio of phycocyanin extract was obtained when fresh biomass was used as raw material. The crude extract was purified by membrane process using microfiltration and ultrafiltration. Membrane of pore sizes 5 μm, at feed flow rate of 150 mL min⁻¹, permeate flux of 58.5 L h⁻¹ m⁻² was selected for coarse filtration and membrane with pore size 0.8/0.2 μm at the flow rate of 100 mL min⁻¹, permeate flux of 336 L h⁻¹ m⁻² was selected for fine filtration, giving phycocyanin recovery of 88.6% and 82.9%, respectively. For ultrafiltration, membrane with MWCO at 50 kDa, 69 kPa and 75 mL min⁻¹ of flow rate with a mean permeate flux 26.8 L h⁻¹ m⁻² and a retention rate of 99% was found to be optimal. Under these filtration conditions, food grade phycocyanin with the purity around 1.0 containing c-phycocyanin as the major component was obtained.     

Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the host's central carbon metabolism.To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, ¹³C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the ¹³C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol_ATP g_CDW⁻¹ h⁻¹ for the reference strain to 45 mmol_ATP g_CDW⁻¹ h⁻¹ for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5 ± 0.1 mmol g_CDW⁻¹ h⁻¹, hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7 g_CDW mmol_ATP⁻¹ for the reference strain to 4.9 g_CDW mmol_ATP⁻¹ for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of microbial hosts and fermentation conditions.     

Large catalase based bioelectrode for biosensor application

A large catalase (CAT) (Mr ~ 90 kDa), immobilized on multiwalled carbon nanotubes—Nafion^® (MWCNT-NF) matrix and encapsulated with polyethylenimine (PEI) on glassy carbon electrode (GCE), showed a pair of nearly reversible cyclic voltammetric peaks for Fe^(III)/Fe^(II) couple with formal potential of about −0.45 V (vs. Ag/AgCl electrode at pH 7.5). PEI significantly reduced the charge transfer resistance and stabilized the bioelectrode through electrostatic interaction. The electron transfer rate constant and surface coverage of the immobilized CAT were 1.05 ± 0.2 s⁻¹ and 2.1 × 10⁻¹⁰ mol cm⁻², respectively. Studies on electrocatalytic activity and kinetics of GCE/MWCNT-NF/CAT/PEI for hydrogen peroxide (H₂O₂) showed the apparent Michaelis-Menten constant of 3 mM, linear response in the range of 10 μM to 5 mM, response time of ~ 2 s for steady state current, and detection limit of ~ 1 μM. A high operational and storage stability was also demonstrated for the bioelectrode. Hence, the direct electrochemistry of the large catalase and its potential biosensor application have been established through this investigation.     

Integrated system investigating shear-mediated platelet interactions with von Willebrand factor using microliters of whole blood

We report an integrated platelet translocation analysis system that measures complex dynamic platelet-protein surface interactions in microliter volumes of unmodified anticoagulated whole blood under controlled fluid shear conditions. The integrated system combines customized platelet-tracking image analysis with a custom-designed microfluidic parallel plate flow chamber and defined von Willebrand factor surfaces to assess platelet trajectories. Using a position-based probability function that accounts for image noise and preference for downstream movement, outputs include instantaneous and mean platelet velocities, periods of motion and stasis, and bond dissociation kinetics. Whole blood flow data from healthy donors at an arterial shear rate (1500 s⁻¹) show mean platelet velocities from 8.9 ± 1.0 to 12 ± 4 μm s⁻¹. Platelets in blood treated with the antiplatelet agent c7E-Fab fragment spend more than twice as much time in motion as platelets from untreated control blood; the bond dissociation rate constant (k_off) increases 1.3-fold, whereas mean translocation velocities do not differ. Blood from healthy unmedicated donors was used to assess flow assay reproducibility, donor variability, and the effects of antiplatelet treatment. This integrated system enables reliable, rapid populational quantification of platelet translocation under pathophysiological vascular fluid shear using as little as 150 μl of blood.     

The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques

Nisin, a peptide antibiotic, efficiently kills bacteria through a unique mechanism which includes inhibition of cell wall biosynthesis and pore formation in cytoplasmic membranes. Both mechanisms are based on interaction with the cell wall precursor lipid II which is simultaneously used as target and pore constituent. We combined two biosensor techniques to investigate the nisin activity with respect to membrane binding and pore formation in real time. Quartz crystal microbalance (QCM) allows the detection of nisin binding kinetics. The presence of 0.1 mol% lipid II strongly increased nisin binding affinity to DOPC (k_D 2.68 × 10^− 7 M vs. 1.03 × 10^− 6 M) by a higher association rate. Differences were less pronounced while using negatively charged DOPG membranes. However, lipid II does not influence the absolute amount of bound nisin. Cyclic voltammetry (CV) data confirmed that in presence of 0.1 mol% lipid II, nanomolar nisin concentrations were sufficient to form pores, while micromolar concentrations were necessary in absence of lipid II. Both techniques suggested unspecific destruction of pure DOPG membranes by micromolar nisin concentrations which were prevented by lipid II. This model membrane stabilization by lipid II was confirmed by atomic force microscopy. Combined CV and QCM are valuable to interpret the role of lipid II in nisin activity.     

Transferrin receptor-1 iron-acquisition pathway — Synthesis,kinetics, thermodynamics and rapid cellular internalization of a holotransferrin–maghemite nanoparticle construct

Background

Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy?

Methods

Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6 nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe₂) by amide bonds (TFe₂–NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe₂–NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy.

Results

In-vitro, R1 interacts with TFe₂–NP with an overall dissociation constant K_D = 11 nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe₂–NP interacts with R1 in 50 μs: second-order rate constant, k₁ = 6 × 10¹⁰ M^− 1 s^− 1; first-order rate constant, k_− 1 = 9 × 10⁴ s^− 1; dissociation constant, K_1d = 1.5 μM. In the second step, the protein/protein adduct undergoes a slow (10,000 s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe₂. In HeLa cells, TFe₂–NP is internalized in the cytosol in less than 15 min.

Conclusion

This is the first time that a nanoparticle–transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin.

General significance

TFe₂–NP behaves as free TFe₂ and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.     

Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA

Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin-iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb³⁺) stoichiometry and an affinity of K =( 2 ± - 1 )× 10¹¹ M^− 2 was determined. Pch-Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb³⁺) and Tb³⁺ (acceptor). Pch-Tb binds to the Pch-Fe outer membrane transporter FptA with a dissociation constant (K_d) of 4.6 μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch.     

Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 °C, at an operating potential of +0.4 V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol⁻¹ was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n = 4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R² = 0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20 mM, with those determined spectrophotometrically (R² = 0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10⁶ cells min) based on a 24-h period in culture.     

Kinetics and bioreactor studies of immobilized invertase on polyurethane rigid adhesive foam

A new support, polyurethane rigid adhesive foam (PRAF), which can be used to cover internal surface of metallic tubes, was used to immobilize invertase for application in an enzymatic bioreactor. The kinetic parameters were: K_m - 46.5 ± 1.9 mM (PRAF-invertase) and 61.2 ± 0.1 mM (free enzyme) and V_max 42.0 ± 4.3 U/mg protein/min (PRAF-invertase) and 445.3 ± 24.0 U/mg protein/min (free invertase). The PRAF-invertase derivative maintained 50.1% of initial activity (69.17 U/g support) for 8 months (4 °C) and was not observed microbial contamination. The bioreactor showed the best production of inverted sugar syrup using up-flow rate (0.48 L/h) with average conversion of 10.64 ± 1.5% h⁻¹ at feeding rate (D) of 104 h⁻¹. The operational inactivation rate constant (k_opi) and half-life were 1.92 × 10⁻⁴ min⁻¹ and 60 h (continue use). The PRAF spray support looks promising as a new alternative to produce immobilized derivatives on reactor surfaces.