Characterisation of chitosan and its films for tissue engineering

Abstract

Chitosan is a renewable polymer produced from a waste product of the seafood industry. It has been seen as useful for a range of applications due to its inherent properties. It is antifungal, antibacterial, biodegradable, biocompatible and has low immunogenicity which makes it attractive specifically for biomedical applications. Examples of chitosan's possible applications include bioadhesive films, stem cell growth substrates and drug delivery agents. Chitosan is produced from the N-deacetylation of chitin. Chitin is the second most abundant polysaccharide in the world (by volume after cellulose) and is synthesized by many organisms which results in it being readily available, inexpensive and abundant. Its natural occurrence includes the shells of arthropods such as shrimps, crabs and the cell walls of yeasts. Unfortunately, due to its natural origin and the variation in processing conditions, chitosan is plagued by batch-to-batch variations which affect its widespread utilization. It thus requires appropriate characterization to allow exploitation of its inherent properties. The molecular structure of chitosan includes varying proportions of Dglucosamine and N-acetyl- D-glucosamine monomer units. The degree of acetylation (DA) is defined as the fraction of N-acetyl- D-glucosamine and the distribution of DAs is defined as its variation between polymer chains in a given sample. Although it has been well documented that a distribution of DAs exists (not all chitosan chains have the same DA), this is often overlooked. Therefore common characterization of chitosan is often incomplete and only takes into account one of the average DAs and neglects the distributions of DAs. The complexity and importance of the distribution of DAs had been revealed recently through a coupling of size-exclusion chromatography (SEC) with 1H NMR spectroscopy; however, it had not been measured before the work in this PhD. To allow an accurate characterization of polymers in solution, a true solution must be obtained. Unfortunately, the dissolution of chitosan is often overlooked. Utilizing capillary electrophoresis in the critical conditions (CECC), solution- and solid-state NMR spectroscopy, the dissolution of chitosan was probed. Obtaining a true chitosan solution has been proven to be challenging even with commonly used aqueous solvents. Aqueous AcOH which is most commonly used was seen to dissolve chitosan inefficiently compared to aqueous HCl. However, significant deacetylation was seen in chitosan dissolved in aqueous HCl and kept at high temperatures for prolonged periods of time. The standard for polymer size analysis, SEC, was shown to detect aggregation of the chitosan chains in the SEC eluent. Furthermore, comparisons of the average DA obtained with solution-state compared to solidstate NMR spectroscopy gave evidence of a clear bias in the characterization due to incomplete dissolution. This is extremely significant as chitosan is often characterized with solution-state NMR spectroscopy. The dissolution was concluded to be complex and a compromise is necessary in allowing a more complete dissolution and minimal deacetylation. However, for routinely measured average DA values, measurements should be undertaken in the solid state. To allow a more comprehensive characterization of chitosan composition, methods were developed in this PhD using free solution capillary electrophoresis in the critical conditions (CE-CC). CE-CC is a separation method and therefore is able to yield distributions. Complex polymers can have distributions of various parameters including composition, branching, end groups and molar mass. For chitosan, CE-CC separates by composition (or degree of acetylation). Capillary electrophoresis has been proven to be a robust technique for the separation and characterization of both natural and synthetic polymers. A method was developed to calculate dispersities from distributions obtained with CE-CC analogous to the calculation of dispersity from molar mass distributions determined by size-exclusion chromatography (SEC). Using a ratio of moments, the dispersity of electrophoretic mobility and composition distributions were obtained. The dispersity values represented either the heterogeneity of branching or composition of the complex polymers. This resulted in further characterization of complex polymers based on their composition or architecture. The dispersity values allowed the quantification and numerical representation of the heterogeneity. This allowed comparisons and trends to be quantified between samples. In the further analysis of chitosan, improvements in the separation were sought. This included changing the counter-ion of the buffer during the CE separation from sodium to lithium. Lithium showed trends of greater selectivity and combining these results with previous work in reducing the adsorption (lower pH and higher temperature) allowed a more accurate separation. The dispersity was then calculated for a larger range of chitosan samples and both distributions of electrophoretic mobilities and composition distributions were obtained. A trend was seen in which the dispersity first increased with the average DA and then began to reduce. Using the correlation between composition and mobility allowed composition distributions to be obtained for chitosan for the first time. This was the first determination of composition distributions and of their corresponding dispersity values for a statistical copolymer. The results identified chitosan samples with very similar measured average DA values having significantly different dispersity values. These results confirm the inaccuracy of characterizing chitosan by only through its average DA. Finally, to improve the use of chitosan for tissue engineering, regenerative medicine and other biomedical applications it was important to ensure low immunogenicity especially in the application of implantation. Poly(ethylene glycol), PEG, and the peptide RGDS was grafted onto the surface of chitosan and the chemical reaction was monitored using CE. The robustness of CE allows samples to be injected without sample preparation and allows it to be used effectively in the analysis of chemical reactions. The films were then characterized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) and the grafting of PEG onto the surface of the chitosan film was validated. Previous cell attachment studies showed that the proliferation of cells occurred in specific regions. This was likely due to an inherent heterogeneity of the chitosan films which could be caused by incomplete dissolution and aggregation seen in the dissolution studies. To probe this heterogeneity of the chitosan films and powder, advanced solid-state NMR spectroscopy measurements were undertaken. The analysis compared the mobile and rigid fractions of chitosan. The results suggested similar behavior in both fractions, however, gave evidence of possible orientation of the acetyl group away from the backbone. The permeability of the films to small molecules was also tested and confirmed. In summary, the dissolution of chitosan was seen to be complex and currently used methods were either deemed inefficient in the dissolution or conversely caused degradation. A new method was developed to numerically represent the heterogeneity of composition or branching and this was tested with various complex polymer samples including chitosan. Further development of this method allowed composition distributions of a statistical copolymer (chitosan) to be obtained for the first time and the heterogeneity of composition to be obtained. New low immunogenicity films were produced by the grafting of poly(ethylene glycol) onto the surface of chitosan films and the grafting process was monitored by CE. The grafting was validated and the permeability and heterogeneity of chitosan films were also probed. Future work should involve probing the dissolution of chitosan with ionic liquids, applying the calculation of dispersity of both distributions of electrophoretic mobility and composition distributions to a broader range of polyelectrolytes, further improving the selectivity of the separation of chitosan and testing the biocompatibility of PEG grafted chitosan films. The methods developed in this thesis will enable chitosan to reach its potential for various applications ranging from tissue regeneration, through bioplastics to drug delivery.

Date of Award	2016
Original language	English