Faaizah Khan

Nanomedicine and its potential in diabetes research and practice

Nanomedicine involves measurement and therapy at the level of 1–100 nm. Although the science is still in its infancy, it has major potential applications in diabetes. These include solving needs such as non‐invasive glucose monitoring using implanted nanosensors, with key techniques being fluorescence resonance energy transfer (FRET) and fluorescence lifetime sensing, as well as new nano‐encapsulation technologies for sensors such as layer‐by‐layer (LBL) films. The latter might also achieve better insulin delivery in diabetes by both improved islet encapsulation and oral insulin formulations. An ‘artificial nanopancreas’ could be an alternative closed‐loop insulin delivery system. Other applications of nanomedicine include targeted molecular imaging in vivo (e.g. tissue complications) using quantum dots (QDs) or gold nanoparticles, and single‐molecule detection for the study of molecular diversity in diabetes pathology. Copyright

Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein

We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO(3) as templates and encapsulated in alternating nano-layers of poly-L-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules. Addition of glucose increased the mean lifetime of GBP-badan by a maximum of approximately 2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (approximately 0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (approximately 3.1 ns) representing the closed form with bound glucose and where the lobes of GBP have closed round the dye creating a more hydrophobic environment. FLIM demonstrated that increasing glucose increased the fractional proportion of the long-lifetime component. We conclude that fluorescence lifetime-based glucose sensing using GBP encapsulated with nano-engineered layer-by-layer films is a glucose monitoring technology suitable for development in diabetes management.

Fluorescence intensity- and lifetime-based glucose sensing using an engineered high-Kd mutant of glucose/galactose-binding protein.

We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213R/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on the addition of glucose, with a binding constant (K(d)) of 11 mM, an operating range of approximately 1-100 mM, and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that the GBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sensor for in vivo glucose monitoring in diabetes.

A fluorescence lifetime-based fibre-optic glucose sensor using glucose/galactose-binding protein

Alternative, non-electrochemistry-based technologies for continuous glucose monitoring are needed for eventual use in diabetes mellitus. As part of a programme investigating fluorescent glucose sensors, we have developed fibre-optic biosensors using glucose/galactose binding protein (GBP) labelled with the environmentally sensitive fluorophore, Badan. GBP-Badan was attached via an oligohistidine-tag to the surface of Ni-nitrilotriacetic acid (NTA)-functionalized agarose or polystyrene beads. Fluorescence lifetime increased in response to glucose, observed by fluorescence lifetime imaging microscopy of the GBP-Badan-beads. Either GBP-Badan agarose or polystyrene beads were loaded into a porous chamber at the end of a multimode optical fibre. Fluorescence lifetime responses were recorded using pulsed laser excitation, high speed photodiode detection and time-correlated single-photon counting. The maximal response was at 100 mM glucose with an apparent K(d) of 13 mM (agarose) and 20 mM (polystyrene), and good working-day stability was demonstrated. We conclude that fluorescence lifetime fibre-optic glucose sensors based on GBP-Badan are suitable for development as clinical glucose monitors.

Cadmium bound to metal rich granules and exoskeleton from Gammarus pulex causes increased gut lipid peroxidation in zebrafish following single dietary exposure

There has been a growing interest in establishing how the sub-cellular distribution of metals in macro-invertebrate prey affects metal trophic bioavailability and toxicity. In this study, the crustacean Gammarus pulex was exposed to 300mugCdl(-1) spiked with (109)Cd for 13 days, from which the two principal metal containing sub-cellular fractions, the metallothionein-like protein (MTLP) and the metal rich granule and exoskeleton (MRG+exo) were isolated. These fractions were produced at equal metal content, incorporated into gelatin and fed to zebrafish as a single meal; assimilation efficiency (AE), carcass and gut tissue metal concentrations and gut lipid peroxidative damage measured as malondialdehyde (MDA) were assessed. The AE of cadmium bound to the MTLP fraction was 32.1+/-5.6% which was significantly greater than the AE of MRG+exo bound Cd, 13.0+/-2.1% (p<0.05). Of the metal retained by the fish at 72h post-feeding, 94% of MTLP-Cd had been incorporated into the carcass, whereas a significant proportion (46%) of MRG+exo-Cd, although assimilated, appeared to remain associated with intestinal tissue. However, this did not translate into a gut tissue concentration difference with 6.8+/-1.2ngCdg(-1) in fish fed MTLP-Cd compared to 9.5+/-1.4ngCdg(-1) in fish fed MRG+exo fraction. Both feeds led to significantly increased MDA levels compared to the control group (gelatin only feed), but MRG+exo feed caused significantly more oxidative damage than the MTLP feed (p<0.01). Thus, MTLP-Cd is more bioavailable than the cadmium bound to granules and exoskeleton, but it was the latter fraction, largely considered as having limited bioavailability, that appeared to exert a greater localised oxidative injury to the digestive tract of zebrafish.

Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.

Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine.

Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.

A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration.

Both lung disease and elevation of blood glucose are associated with increased glucose concentration (from 0.4 to ~4.0 mM) in the airway surface liquid (ASL). This perturbation of ASL glucose makes the airway more susceptible to infection by respiratory pathogens. ASL is minute (~1 μl/cm2) and the measurement of glucose concentration in the small volume ASL is extremely difficult. Therefore, we sought to develop a fluorescent biosensor with sufficient sensitivity to determine glucose concentrations in ASL in situ. We coupled a range of environmentally sensitive fluorophores to mutated forms of a glucose/galactose-binding protein (GBP) including H152C and H152C/A213R and determined their equilibrium binding properties. Of these, GBP H152C/A213R–BADAN (Kd 0.86 ± 0.01 mM, Fmax/F0 3.6) was optimal for glucose sensing and in ASL increased fluorescence when basolateral glucose concentration was raised from 1 to 20 mM. Moreover, interpolation of the data showed that the glucose concentration in ASL was increased, with results similar to that using glucose oxidase analysis. The fluorescence of GBP H152C/A213R–BADAN in native ASL from human airway epithelial cultures in situ was significantly increased over time when basolateral glucose was increased from 5 to 20 mM. Overall our data indicate that this GBP is a useful tool to monitor glucose homoeostasis in the lung.

Nanomedicine in diabetes management: where we are now and where next

‘Nanos’ is the Greek for dwarf and, in its broadest sense, nanotechnology involves making or measuring things on a very small scale. When this technology is applied to the problems of medicine it is called ‘nanomedicine’. As with many buzz words in bioscience (e.g., biotechnology, molecular medicine), nanotechnology and nanomedicine are somewhat loosely and arbitrarily defined and use aspects of several sciences that are not totally new [1]. How small must a structure be to be included in nanomedicine, for example? Although a nanometer (nm) is 10 meters, by convention nanotechnology has so far mostly dealt with structures from 1 to 100 nm in size – larger than an atom but smaller than a cell.