Design of Rheological Measurements for Rapidly Gelling Polysaccharides on Exposure to External Cross-Linkers

This research project focused on a range of gel forming polysaccharides, including sodium alginate pectin and gellan gum. All of these materials have ability to form a gel responding to different stimuli, such as pH and crosslinking ions. Their capability to undergo sol-gel transition in presence of mono or divalent cations can often occur in seconds making it particularly difficult to measure the gelation in real time. Therefore, the aim of the work presented in this thesis was to design a new technique that allows commercially available rheological equipment to monitor the gelation progress of these materials.

The first part of study involved the design a new method to measure the gelation of alginate and pectin in situ on exposure to an external source of calcium ions. Direct mixing of alginate or low methoxy pectin with divalent cations such as Ca2+ generally produces heterogeneous gels that form almost instantaneously. Therefore, it is particularly difficult to measure the rheological properties of this gelation event due to the rapid gelation kinetics. In this study the gelation progress was measured on exposure to three different concentrations of CaCl2 and gel dissolution time was measured by removing the crosslinking Ca2+ from freshly formed alginate and pectin gels by exposure to calcium chelators. The modification of the rheometer to facilitate these measurements used a petri dish attached to the lower plate of the rheometer, into which a piece of filter paper submerged with calcium chloride solutions (50, 100 and 200 mM) was placed. On top of the filter paper dialysis membrane (MWCO 14000) was placed as a barrier to prevent the filter paper imbibing polysaccharide samples. Samples of alginate and pectin 4% w/w were loaded on to the membrane and small deformation oscillatory measurements of elastic modulus (G′) and viscous modulus (Gʺ) were taken in the linear viscoelastic region, to monitor the gelation as a function of time. Once the gelation was complete the filter paper was removed and replaced with filter paper immersed with calcium chelating agents (500 mM of EDTA and sodium citrate (Na citrate)) to degrade the gel in situ. The results demonstrated that this technique was suitable for analysing the external gelation of alginate and pectin with a sharp increase in G′ in the first three minutes which then plateaued over the remainder of the test. It was also shown that gel stiffness reduced to a greater extent on exposure to EDTA compared with Na citrate. This method is not only suitable for measuring rapid gelation kinetics on exposure to cross-linkers, but has potential applications in modelling the in situ gelation behaviour in simulated physiological environments.

The second part of the study investigated using the method developed for the alginate and pectin gelation for the in situ gelation of gellan gum under simulated physiological conditions using different types of simulated body fluids, simulated wound fluid (SWF), artificial saliva (AS), artificial lacrimal fluid (LF) and artificial gastric fluid (GF), measuring the gelation of gellan gum solutions (0.25, 0.5, 0.75 and 1% w/w) in response to ionic crosslinking and acidic pH.

The results showed that gellan made the stiffest gels with GF, followed by LF and SWF (which were of similar stiffness to each other) and the weakest gel was formed with AS. The results indicate that this method is not only of use to measure rapid gelation kinetics on external exposure to cross-linkers but could find application in designing bioresponsive delivery systems in the food, pharmaceutical and biomedical industries.

The final section of this thesis focused on the design and proof of concept of a new device that was 3D printed using Acrylonitrile Butadiene Styrene polymers (ABS), termed a rheo-dissolution cell. This was investigated to overcome the limitations of the petri dish method used in the initial studies. The cell was designed to contain a reservoir capable of holding crosslinking solutions and dissolution media allow with sampling ports to be able evaluate rheology measurements of in situ gel forming systems while simultaneously measuring release of molecules loaded into the gel. On the top of the reservoir a retractable stainless steel mesh was used as the lower plate of the rheometer which allowed the polysaccharide samples to be in contact with the fluid in the reservoir during the gelation and gel dissolution. Proof of concept was determined using 1% w/w alginate solution loaded with 50 mg of methylene blue. Rheological measurements were performed with CaCl2 in the rheo-dissolution reservoir to initiate gelation. Once the gels were formed gel dissolution was initiated by replacing the CaCl2 with calcium chelating agents EDTA and Na citrate. During both of these processes samples from the reservoir were removed and analysed for methylene blue release. The results showed the device was capable of allowing the formation strong gels on exposure to CaCl2 and gel dissolution when the formed gels were exposed to the calcium chelators. It was also found that mesh opening size was an important factor in the final gel strength and subsequent gel dissolution time. The strongest gels were formed when the mesh had a large opening area and the chelating of calcium was faster with EDTA than Na citrate. Methylene blue was detected in the reservoir during both gelation and gel dissolution with gel strength appearing to be an important factor on the quantity released gels increased. This study indicated that the rheo-dissolution cell has the potential to be used as a model system for measuring rheological changes and release rate of loaded drugs in gel forming formulations simultaneously by a simple modification of a commercially available rheometer.