Dave E. Dunstan

Injectable chitosan hydrogels for localised cancer therapy

The pace of development of delivery systems that could target drugs to specific body sites and control the release of drugs for prolonged periods of time have been steady though slow. Till a decade ago, mostly microspheres or nanoparticles were widely studied and applied in cancer treatment. However, due to shortcomings of these systems, there has been a surge in interest for in situ hydrogels. This review focuses on the current use of injectable in situ chitosan hydrogels in cancer treatment. Formulation protocols for in situ hydrogel systems, their cytotoxic properties, loading and in vitro release of drugs, their effect on cell growth in vitro and inhibition of tumor growth in vivo using mouse models, and future directions to enhance this technology are discussed.

Characterisation of xanthan gum solutions using dynamic light scattering and rheology

Abstract The ‘weak gel’ formation mechanism of xanthan gum solutions has been examined using rheological and light scattering methods. Significant differences are observed between the critical overlap concentration ( c ∗ ) measured using both techniques. The effect of shear on c ∗ for semi-flexible molecules such as xanthan is demonstrated. The approximate theories of Doi and Edwards (1984) . The Theory of Polymer Dynamics. Oxford: Clarendon Press) for rigid rods and De Gennes (1979) . Scaling Concepts in Polymer Solutions. London: Cornell University Press) for random coils are used to explain the differences observed between rheological and light scattering measurements of c ∗ . A second concentration regime parameter due to aggregation, c ∗∗ , is observed in dynamic light scattering results (DLS). The parameter c ∗∗ has been previously observed for xanthan systems. Light scattering measurements in various electrolytes suggest that the use of DLS is able to distinguish between the transition from truly dilute behaviour to semi-dilute behaviour ( c ∗ ) and the critical concentration for aggregation ( c ∗∗ ).

The effects of shear flow on protein structure and function.

Protein molecules are subjected to potentially denaturing fluid shear forces during processing and in circulation in the body. These complex molecules, involved in numerous biological functions and reactions, can be significantly impaired by molecular damage. There have been many studies on the effects of hydrodynamic shear forces on protein structure and function. These studies are reviewed and the implications to bioprocessing and pathophysiology of certain diseases are discussed.

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers --> monomers --> partially unfolded monomers --> oligomeric aggregates --> fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.

Shear flow promotes amyloid-β fibrilization

The rate of formation of amyloid fibrils in an aqueous solution of amyloid-beta (Abeta) is greatly increased when the solution is sheared. When Abeta solution is stirred with a magnetic stirrer bar at 37 degrees C, a rapid increase in thioflavin T fluorescence is observed. Atomic Force Microscopy (AFM) images show the formation of aggregates, the growth of fibrils and the intertwining of the fibrils with time. Circular dichroism (CD) spectroscopy of samples taken after stirring shows a transition from random coil to alpha-helix to beta-sheet secondary structure over 20 h at 37 degrees C. The fluorescence, AFM and CD measurements are all consistent with the formation of amyloid fibrils. Quiescent, non-stirred solutions incubated at 37 degrees C showed no evidence of amyloid formation over a period of 3 days. Couette flow was found to accelerate the formation of amyloid fibrils demonstrating that the primary effect of stirring is not mixing but shearing. Only very small shear forces are applied to individual molecules in our experiments. Simple calculation suggests that the force is too small to support a hypothesis that shearing promotes partial unfolding of the protein as is observed.

Rheology of dextran solutions

Commercial dextran samples of molecular weight up to 2 million and concentration up to 30 wt.% in aqueous solutions show Newtonian viscosity behaviour. Plot of shear viscosity as a function of concentration for the 2 million Mw gives a very high critical overlap concentration of approximately 8 wt.%, while the viscosity at 25 wt.% shows a very small molecular weight dependence, in sharp contrast to findings for flexible polymers in the semi-dilute region. All these results point to the conclusion that the dextran molecules are less stiff than most carbohydrates and are highly branched, so that the molecules are in a highly compact configuration in aqueous solution.

Do stable nanobubbles exist in mixtures of organic solvents and water

Several recent papers have described the existence of stable nanobubbles in bulk, which is surprising given that the high curvature of these bubbles is expected to place such bubbles under a high pressure and therefore lead to rapid dissolution. Here, we investigate the possible existence of nanobubbles in mixtures of water plus an organic solvent using both static and dynamic light scattering and infrared spectroscopy. The mixing of solvents was designed to introduce nanobubbles into bulk solution via supersaturation of the solution. The solutions scatter light for a long period (days) after mixing, which is consistent with the formation of nanoscale objects, but we show that these scattering objects originate from water-insoluble impurities in the organic solvents. Our results are inconsistent with the presence of gas nanobubbles in bulk solution: Degassing the solutions, either before or after mixing, has a minimal effect on the scattering, and purification of the organic solvent before mixing reduces the scattering after mixing. Therefore, previous reports of nanobubbles based on scattering experiments should be reconsidered with the hypothesis that the scattering objects are not actually gaseous.

Shear-induced deformation of bovine insulin in Couette flow.

We have applied a uniform, shear-driven flow field (Couette flow) to study the effect of shear on the structure and conformation of aqueous bovine insulin, in situ and in real time, using intrinsic Tyr fluorescence and circular dichroism (CD) spectroscopy. The morphology of post-shear insulin samples was analyzed using atomic force microscopy (AFM). Both fluorescence and CD data show a shear-dependent deformation of bovine insulin in Couette flow. The shear effect is more pronounced with increasing shear rate. AFM images show large aggregates for insulin samples sheared at 200 and 400 s(-1), whereas samples sheared at 600 s(-1) contained fibrillar forms. We hypothesize that helical segments unfold upon extensional strain in the deformation flow field, resulting in unstructured, aggregation-prone insulin molecules. The occurrence of rotational diffusion in the direction of flow facilitates the coalescence of deformed insulin molecules into oligomeric aggregates. The size of the insulin aggregates diminished with increasing shear rate. This shows that the deformation cycle in fast flow fields retards the formation of large aggregates and promotes the ordering of deformed insulin molecules into the more stable fibrillar forms.

Shear-induced structure and mechanics of β-lactoglobulin amyloid fibrils

Alzheimer first identified amyloid plaques during autopsies of the brains of dementia patients in 1905. Considerable effort has since been directed to understanding the composition, structure and formation mechanism of amyloid fibrils. A variety of proteins have now been shown to self-assemble into amyloid fibrils. Furthermore, the association between misfolded proteins and a wide range of diseases is now established. Here we examine the effect of shear on the formation, structure and mechanical properties of amyloid fibrils. Atomic force microscopy is used to both image and measure the nanomechanical properties of β-lactoglobulin (β-Lg) amyloid fibrils generated in controlled (Couette) and uncontrolled shear flows (stirring). Controlled shear-induced fibrils with ‘beaded’ morphologies have lower mechanical strengths than fibrils formed in uncontrolled shear which show twisted ribbon like morphologies. The mechanical strengths of amyloid fibrils are determined by fibril structure and vary depending on the type of shear exposure during in vitro formation. These findings may have implications regarding the different toxicities of amyloid fibrils and linear aggregates that form in vivo.

Rheological characterisation of ‘weak gel’ carrageenan stabilised milks

Abstract The use of a controlled stress rheometer to characterise the rheology of weak gel behaviour of milk containing carrageenan is described. The interaction of carrageenan with the protein in milk produces a long-range network structure where the physical properties are dependent on the type of carrageenan used. With the use of a sensitive controlled stress rheometer, it was possible to characterise the rheological properties of these weak gels and differentiate between samples using both large deformation techniques, and small amplitude oscillatory measurements. Large deformation tests enabled the gels to be qualitatively compared based on an apparent yield stress, ‘structure point’, apparent shear viscosity and degree of hysteresis. Small amplitude oscillatory rheometry was applied to make comparisons from measurements that did not exceed the linear viscoelasticity of the system. Both the frequency and time dependence of the rheological properties were measured to characterise final gel properties and the kinetics of network development. Both large deformation rheology and small amplitude oscillatory rheology have been effective in providing quantitative and qualitative comparisons between the rheological properties of samples containing weak gel networks.