Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate.

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.

Crisis resolution: consumer, family and referrer perspectives on care.

AIM: To systematically assess the service satisfaction of consumers, their families and referrers with crisis resolution (CR). METHODS: Consecutive consumers discharged after receiving CR over a five-week period were potentially eligible for participation, together with their family and referrer (broadly defined). Structured telephone interviews were conducted and involved forced-choice questions assessing global satisfaction and satisfaction with specific aspects of care, plus two open-ended questions. RESULTS: Participants were 75 consumers, 22 family and 16 referrers. High levels of satisfaction were seen for all participants for both global (86-96%) and most specific aspects of care (>75%). If consumers were dissatisfied with their overall care, they were significantly more likely to be aged 25-34 years of age. High levels of agreement among raters were found for global satisfaction (>85%) and most specific aspects of care (>70%), which provides some level of reassurance for staff. Open-ended questions showed that having effective treatment of sufficient duration and staff manner were most important to participants. CONCLUSION: High levels of satisfaction and agreement were found among consumers, family and referrers with CR. Open-ended questions identified which issues matter the most to key stakeholders, which may have implications for service evaluation tools.

Thermoresponsive Gels.

Thermoresponsive gelling materials constructed from natural and synthetic polymers can be used to provide triggered action and therefore customised products such as drug delivery and regenerative medicine types as well as for other industries. Some materials give Arrhenius-type viscosity changes based on coil to globule transitions. Others produce more counterintuitive responses to temperature change because of agglomeration induced by enthalpic or entropic drivers. Extensive covalent crosslinking superimposes complexity of response and the upper and lower critical solution temperatures can translate to critical volume temperatures for these swellable but insoluble gels. Their structure and volume response confer advantages for actuation though they lack robustness. Dynamic covalent bonding has created an intermediate category where shape moulding and self-healing variants are useful for several platforms. Developing synthesis methodology-for example, Reversible Addition Fragmentation chain Transfer (RAFT) and Atomic Transfer Radical Polymerisation (ATRP)-provides an almost infinite range of materials that can be used for many of these gelling systems. For those that self-assemble into micelle systems that can gel, the upper and lower critical solution temperatures (UCST and LCST) are analogous to those for simpler dispersible polymers. However, the tuned hydrophobic-hydrophilic balance plus the introduction of additional pH-sensitivity and, for instance, thermochromic response, open the potential for coupled mechanisms to create complex drug targeting effects at the cellular level.

In vivo study of a polymeric glucose-sensitive insulin delivery system using a rat model.

This study assesses the feasibility of an intraperitoneal (IP) implantable closed-loop insulin delivery device in rats, that delivers insulin via a glucose-sensitive material such that blood glucose (BG) levels are adjusted automatically to within normal tolerances. A gateway layer of this gel governs the output of insulin from an insulin reservoir device for IP implant. The performance of the system was compared over time in diabetic rats with a control system using oral glucose challenges and daily assessments of BG and body weight. The automated response of the active system was quantified using IP multiple dose injection (MDI) results in the same rat model. Successful control was found for the device containing active gel when assessed daily and when challenged with large glucose doses. This was not found when comparing an inactive gel analog as a control. The regimen was quantified by comparison with the informative MDI study. The device was well tolerated and might operate to further advantage when vascular omentum grows into the perforated front of the device. The successful device must have been outputting approximately 0.5 U/kg/h basal with 2 U/kg boosts in order to match the demand of the challenges. However, the device eventually exhausts and a refill mechanism needs to be devised in future models.

Glucose-sensitive gel rheology of dextran-concanavalin A mixtures suitable for self-regulating insulin delivery.

Aqueous concentrated plain mixtures of dextran and concanavalin A (con A) were examined for their rheological response to glucose for comparison with previously studied partially photopolymerized acrylic derivatives. Non-destructive oscillatory tests were undertaken within the linear viscoelastic range to examine the relationship between the rheometry and the stoichiometry of the interactive materials and to examine rheological parameters as affected by molecular weight, component ratio, temperature and glucose concentrations between 0 and 1% w/w. These simple formulations were studied at 1 and 10 Hz at 0.5% strain at both 20 and 37 degrees C. A second simplified rheological test was undertaken to demonstrate gel-sol reversibility and to produce a measure of equilibria created between these gels and glucose solutions with which they are in contact. This mimics the conditions in which the gel acts as a responsive gateway in the insulin delivery device. It proved that the gels equilibrate with glucose solutions, rather than indiscriminately removing glucose. This is important in terms of producing a delivery device that can respond in a reversible, glucose concentration-dependent manner. The method used for this is capable of relative values only but provides information not obtainable from conventional rheometry.

UV cross-linked dextran methacrylate--concanavalin A methacrylamide gel materials for self-regulated insulin delivery.

In this study, the successful acrylic derivatization of dextran and concanavalin A (con A) to form dextran methacrylate and con A methacrylamide is shown. These derivatized acrylic monomers are then photopolymerized in the presence of a water soluble photoinitiator Irgacure under various conditions to form covalently bonded glucose-responsive gel materials, which undergo a transformation to sol in the presence of free glucose. Rheological data have revealed that as the degree of substitution for dextran methacrylate is increased, a more elastic material is produced due to the increased covalent linkages. Some of these gel systems show negligible component loss in in vitro diffusion experiments used to simulate the behavior of the cross-linked gel, as would be used in a self-regulated insulin delivery device.

Development of a reversed-phase high-performance liquid chromatography method for the analysis of components from a closed-loop insulin delivery system.

A reversed-phase HPLC method has been developed which enables separation of the three components of a closed-loop insulin delivery system, namely concanavalin A methacrylamide (Con A-MA), dextran methacrylate (Dex-MA) and bovine insulin. The analysis of Con A-MA represents a significant challenge due to the formation of multiple conformations on contact with the chromatographic surface and the mobile phase. The extent of conformational change is shown to be dependent on a number of parameters: column temperature, mobile phase pH, contact time with the chromatographic surface, salt type and concentration and the organic modifier. By manipulation of these variables, protein denaturation can be minimised and recovery improved.

The effect of degree of acrylic derivatisation on dextran and concanavalin A glucose-responsive materials for closed-loop insulin delivery.

Formulations of dextran methacrylate (dex-MA) and concanavalin A methacrylamide (con A-MA) were photo-polymerized to produce covalently cross-linked glucose-responsive materials for the basis of a closed-loop insulin delivery device. The viscoelastic properties of these polymerised materials were tested rheologically in the non-destructive oscillatory mode within the linear viscoelastic range at glucose concentrations between 0% and 5% w/w. The degree of acrylic substitution was varied for the dex-MA and con A-MA, and as the formulation glucose concentration was raised, a graded decrease in storage modulus, loss modulus and complex viscosity when compared at 1 Hz was observed for each cross-linked material. Increasing the degree of substitution (DS) of the derivatised dextran produced viscosity profiles at higher values throughout the glucose concentration range. A comparison with non-polymerised mixtures shows similar rheological properties but at much lower values across the chosen glucose concentration range. High-pressure liquid chromatography analyses and in vitro diffusion experiments showed that there were optimum degrees of derivatisation to minimise dex-MA and con A-MA component leach from the material. The in vitro diffusion experiments also showed that differential delivery of insulin in response to glucose was possible with candidate polymerised glucose-responsive formulations, thus highlighting the potential of such a novel glucose-sensitive material to be used as part of implantable closed-loop insulin delivery device.

Rheological characterisation of dextran-concanavalin A mixtures as a basis for a self-regulated drug delivery device.

The rheological characterisation of glucose sensitive mixtures containing dextran and concanavalin A (con A) with and without glucose, was undertaken using oscillatory rheometry at 20 and 37 degrees C so that comparative data could be gathered in the linear viscoelastic (LVE) range. Measurements for a series of mixtures showed that complex viscosity is a function not only of the con A concentration but of the content and molecular weight of the dextran used. The extent of liquefaction on addition of glucose also depended on these factors. The tan delta profiles confirmed the change from semi-solid towards fluid behaviour. This occurs when glucose effects dismantling of the three-dimensional structure of the dextran-con A system by competitive binding to the glucose receptors in the protein. For the mixtures studied, the changes occurred between contents of 0 and 1% (w/w) glucose at 20 and 37 degrees C and form a useful basis for the formulation of a self-regulating delivery device for the control of hyper-and hypoglycaemia in diabetes.

Comparison of micro- and mesoporous inorganic materials in the uptake and release of the drug model fluorescein and its analogues.

The uptake of the three species of the drug model fluorescein (fluorescein sodium salt (FNa), fluorescein free acid (F), and fluorescein diacetate (FDA)) by zeolite NaX and the mesoporous zeotype MCM-41 was investigated as well as their release rates into solutions at pH 7 and pH 4.5. UV/Vis analysis was carried out at a wavelength of 490 nm. Uptakes of the sodium salt of 9 % for zeolite X and 14 % for MCM suggest little penetration of the pores. The use of ethanol as the loading solvent for F resulted in little uptake for both zeolitic materials due to the successful competition of the ethanol for binding sites. Use of acetone (weaker proton acceptor) as loading solvent significantly improved the uptake of F to 17 % and 12 % for zeolite X and MCM, respectively, whilst the uptake of FDA in acetone increased still further to 22 % and 17 % for zeolite X and MCM, respectively. Generally there was a large initial release of the fluorescein analogues from the surface of the zeolites with very little further increase over time. The prescence of an esterase enzyme in the release medium of FDA tripled the release from MCM to 15 % but left the release from zeolite X unaffected at 6 %. The results obtained show that uptake of fluorescein and its analogues is dependent on the loading solvent used, the amount released is influenced by not only the solvent but the pH and the presence of enzymes in the release medium.