Gary G. Adams

Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells.

This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

Polysaccharide drug delivery systems based on pectin and chitosan.

Abstract Chitosans and pectins are natural polysaccharides which show great potential in drug delivery systems. Chitosans are a family of strongly polycationic derivatives of poly-N-acetyl-D-glucosamine. This positive charge is very important in chitosan drug delivery systems as it plays a very important role in mucoadhesion (adhesion to the mucosal surface). Other chitosan based drug delivery systems involve complexation with ligands to form chitosan nanoparticles with can be used to encapsulate active compounds. Pectins are made of several structural elements the most important of which are the homogalacturonan (HG) and type I rhamnogalacturonan (RG-I) regions often described in simplified terms as the “smooth” and “hairy” regions respectively. Pectin HG regions consist of poly-glacturonic acid residues which can be partially methyl esterified. Pectins with a degree of methyl esterification (DM) > 50% are known as high methoxyl (HM) pectins and consequently low methoxyl (LM) pectins have a DM < 50%. Low methoxyl pectins are of particular interest in drug delivery as they can form gels with calcium ion (Ca2 +) which has potential applications especially in nasal formulations.

Insulin‐based regimens decrease mortality rates in critically ill patients: a systematic review

To determine whether treatment with glucose‐insulin‐potassium (GIK), insulin and glucose, or insulin by itself is beneficial in limiting organ damage after acute myocardial infarction (AMI) and reducing mortality and morbidity among critically ill hyperglycaemic patients.

The Delivery of Insulin from Aqueous and Non-Aqueous Reservoirs Governed by a Glucose Sensitive Gel Membrane

A self regulating delivery device, responsive to glucose, has been shown to operate successfully in vitro. This comprises a gel membrane which determines the delivery rate of insulin from a reservoir. The gel consists of a synthetic polysucrose and the lectin, concanavalin A. The mechanism is one of displacement of the branched polysaccharide from the lectin receptors by incoming glucose. The gel loses its high viscosity as a result but reforms on removal of glucose, thus providing the switch controlling the drug diffusion rate. The drug does not require to be chemically modified and thus the device is adaptable to other anti-hyperglycaemic drugs. However, results here indicate that the molecular weight of the solute may be an important parameter. Others include path length, gel formulation and temperature. It had been hypothesised that the reversal might be improved by the use of a non-aqueous reservoir of insulin. However, with the use of insulin, the switching off was found to be superior to that found with other test solutes used in previous studies, irrespective of the reservoir solvent. The advantages in the use of the non-aqueous system include, however, more reproducibility in the magnitude of response and a reduced temperature sensitivity.

Extended Fujita approach to the molecular weight distribution of polysaccharides and other polymeric systems

In 1962 H. Fujita (H. Fujita, Mathematical Theory of Sedimentation Analysis, Academic Press, New York, 1962) examined the possibility of transforming a quasi-continuous distribution g(s) of sedimentation coefficient s into a distribution f(M) of molecular weight M for linear polymers using the relation f(M)=g(s)·(ds/dM) and showed that this could be done if information about the relation between s and M is available from other sources. Fujita provided the transformation based on the scaling relation s=κ(s)M(0.5), where κ(s) is taken as a constant for that particular polymer and the exponent 0.5 essentially corresponds to a randomly coiled polymer under ideal conditions. This method has been successfully applied to mucus glycoproteins (S.E. Harding, Adv. Carbohyd. Chem. Biochem. 47 (1989) 345-381). We now describe an extension of the method to general conformation types via the scaling relation s=κM(b), where b=0.4-0.5 for a coil, ∼0.15-0.2 for a rod and ∼0.67 for a sphere. We give examples of distributions f(M) versus M obtained for polysaccharides from SEDFIT derived least squares g(s) versus s profiles (P. Schuck, Biophys. J. 78 (2000) 1606-1619) and the analytical derivative for ds/dM performed with Microcal ORIGIN. We also describe a more direct route from a direct numerical solution of the integral equation describing the molecular weight distribution problem. Both routes give identical distributions although the latter offers the advantage of being incorporated completely within SEDFIT. The method currently assumes that solutions behave ideally: sedimentation velocity has the major advantage over sedimentation equilibrium in that concentrations less than 0.2mg/ml can be employed, and for many systems non-ideality effects can be reasonably ignored. For large, non-globular polymer systems, diffusive contributions are also likely to be small.

Structure and heterogeneity of gliadin: a hydrodynamic evaluation

A study of the heterogeneity and conformation in solution [in 70% (v/v) aq. ethanol] of gliadin proteins from wheat was undertaken based upon sedimentation velocity in the analytical ultracentrifuge, analysis of the distribution coefficients and ellipsoidal axial ratios assuming quasi-rigid particles, allowing for a range of plausible time-averaged hydration values. All classical fractions (α, γ, ωslow, ωfast) show three clearly resolved components. Based on the weight-average sedimentation coefficient for each fraction and a weight-average molecular weight from sedimentation equilibrium and/or cDNA sequence analysis, all the proteins are extended molecules with axial ratios ranging from ~10 to 30 with α appearing the most extended and γ the least.

Various Non-Injectable Delivery Systems for the Treatment of Diabetes Mellitus

Diabetes mellitus (diabetes) is suffered by more than 180 million people and is responsible for approximately 2.9 million deaths each year. This mortality rate is expected to increase by 50 % in the next decade. Due to the inconvenience of the traditional treatment of diabetes by subcutaneous administration of insulin injection, various attempts are made in the production, purification, formulation and methods of delivery of insulin. However, despite advances in recent years, these attempts have met with limited success. Various alternative routes such as rectal, ocular, nasal, pulmonary and oral have been exploited. The pulmonary route offers great potential for the delivery of polypeptide drugs due to the large surface area for insulin absorption in the respiratory tract. But due to its low bioavailability, oral route is intensely investigated for the insulin delivery. Microencapsulation, as one of the delivery systems utilising oral route, has shown some potential progress in insulin delivery; though it is at an early stage yet it has proved to be quite encouraging providing new less toxic immunosuppressive agents. Microencapsulation may prove to be an attractive delivery system for controlled release of insulin and beneficial for therapeutic, bio-efficient and bio-effective drug delivery. In this review we discuss the possible alternative routes for insulin delivery (ocular, nasal, pulmonary and oral) and advantages and disadvantages of each. Furthermore we consider the different drug delivery strategies available (aerosols, dry powder inhalers, synthetic beta cells, hydrogels and microcapsules) and their current and potential applications with respect to the different insulin delivery routes.

Insulin Delivery Governed by Covalently Modified Lectin-Glycogen Gels Sensitive to Glucose

A glucose‐sensitive gel formulation containing concanavalin A and glycogen has been reported previously. Precipitation resulting from the addition of concanavalin A to glycogen has been documented, but the formation of glucose‐sensitive gels based on lectin‐glycogen interactions is novel and used here in our studies. An improved in‐vitro self‐regulating drug‐delivery system, using covalently modified glucose‐sensitive gels based on concanavalin A and a polysaccharide displacement mechanism, is described. The successful use of the covalently modified gels addresses a problem identified previously where significant leaching of the mitogenic lectin from the gel membranes of non‐coupled gels was encountered.

Extraction, isolation and characterisation of oil bodies from pumpkin seeds for therapeutic use

Pumpkin, a member of the Cucurbitaceae family has been used frequently as functional medicines for therapeutic use. Several phytochemicals such as polysaccharides, phenolic glycosides, 13-hydroxy-9Z, 11E-octadecatrienoic acid from the leaves of pumpkin, proteins from germinated seeds, have been isolated. Here the influence of pH, ionic strength, and temperature on the properties and stability of oil bodies from pumpkin (Cucurbita) were determined with a view to patterning oil body size and structure for future therapeutic intervention. Oil bodies from pumpkin seeds were extracted, isolated, characterised using optical microscopy, zeta potential and particle size distribution obtained. During microscopic analysis, the oil bodies were more intact and in an integrated form at the time of extraction but were ruptured with time. Water extracted oil bodies were spherical for all four layers where cream had larger oil bodies then upper curd. Lower curd and supernatant had considerably smaller size with lower curd densely packed and seemed to be rich in oil bodies than any of the four layers. At pH 3, in the absence of salt, the zeta potential is approximately +30 mV, but as the salt concentration increases, the ζ potential rises at 10 mM but then decreases over the salt range. This trend continues for the upper curd, lower curd and the supernatant and the degree of the reduction (mV) in zeta potential is of the order cream<upper curd<lower curd<supernatant. At pH 7.4, physiological pH, the changes in salt concentration from 10 to 100 mM reduce the zeta potential significantly across all layers such that increased salt concentrations induce negative potentials. Increasing the salt concentrations still further, however, does not make the ζ potential more negative. However, at pH 9 the zeta potential falls from 0 to -50 mV as the salt concentration increases with the largest reduction shown with 100 mM salt. Particle size distribution at increasing pH salt concentration shows the average size distribution of pumpkin seed oil bodies at an increasing pH (3, 7.4 and 9) and salt concentration (0, 10, 50 and 100 mM) across all four layers. The lowest average size distributions are seen at pH 7.4 across all four layers especially within the cream and upper curd layers. At pH 3 and 9, the highest average size distributions are seen in the lower curd and cream layers. Oil bodies can be extracted, isolated and from pumpkins using an aqueous extraction method and may prove to be a useful new source of lipids for application in patterning therapeutics for clinical use.

Ultracentrifuge methods for the analysis of polysaccharides, glycoconjugates, and lignins

Although like proteins, polysaccharides are synthesized by enzymes, unlike proteins there is no template. This means that they are polydisperse, do not generally have compact folded structures, and are often very large with greater nonideality behavior in solution. This chapter considers the relevant analytical ultracentrifuge methodology available for characterizing these and related carbohydrate-based systems and information this methodology supplies, in terms of sizes, shapes, and interactions using a comprehensive range of examples, including glycoconjugates and lignins. The relevance and potential of recent software developments such as SEDFIT-MSTAR, the Extended Fujita algorithm, and HYDFIT are considered.