In vitro hydrolytic degradation of centrifugally spun polyhydroxybutyrate-pectin composite fibres

BACKGROUND: Centrifugal spinning is a novel fibre-forming process that readily permits the incorporation of additives while avoiding the thermal damage often associated with conventional melt spinning. Centrifugal spinning of a viscous solution of poly(3-hydroxybutyrate) (PHB) mixed with pectin was used to fabricate a range of fibres containing different concentrations of this biologically active agent. The influence of this blending on fibre morphology and in vitro degradation in an accelerated hydrolytic model at 70 ?C and pH of 10.6 is reported. RESULTS: Blending influenced the physiochemical properties of the fibres, andthis significantly affected thedegradation profile of both the fibre and its PHB constituent. A greater influence on degradation was exerted by the type of pectin and its degree of esterification than by variations in its loading. CONCLUSION: Centrifugal spinning permits the fabrication of composite fibrous matrices from PHB and pectin. Incorporation of the polysaccharide into the fibres can be used to manipulate degradation behaviour and demonstrates a model for doping of matrices with active biological constituents. The unique features of the centrifugal spinning process, as illustrated by the structure of the fibres and the degradation profiles, suggest possible applications of centrifugally spun biopolymers as wound scaffolding devices and in tissue engineering.

The delivery of bioactive proteins using biodegradable microspheres

In this project, antigen-containing microspheres were produced using a range of biodegradable polymers by single and double emulsion solvent evaporation and spray drying techniques. The proteins used in this study were mainly BSA, tetanus toxoid, F1 and V, Y. pestis subunit vaccines and the cytokine, interferon-gamma. The polymer chosen for use in the vaccine preparation will directly determine the characteristics of the formulation. Full in vitro analysis of the preparations was carried out, including surface hydrophobicity and drug release profiles. The influence of the surfactants employed on microsphere surface hydrophobicity was demonstrated. Preparations produced with polyhydroxybutyrate and poly(DTH carbonate) polymers were also shown to be more hydrophobic than PLA microspheres, which may enhance particle uptake by antigen presenting cells and Peyer's patches. Systematic immunisation with microspheres with a range of properties showed differences in the time course and extent of the immune response generated, which would allow optimisation of the dosing schedule to provide maximal response in a single dose preparation. Both systematic and mucosal responses were induced following oral delivery of microencapsulated tetanus toxoid indicating that the encapsulation of the antigen into a microsphere preparation provides protection in the gut and allows targeting of the mucosal-associated lymphoid tissue. Co-encapsulation of adjuvants for further enhancement of immune response was also carried out and the effect on loading and release pattern assessed. Co-encapsulated F1 and interferon-gamma was administered i.p. and the immune responses compared with singly encapsulated and free subunit antigen.

Biodegradable polymers for the sustained release of antisense nucleic acids

Antisense oligodeoxynucleotides can selectively inhibit individual gene expression provided they remain stable at the target site for a sufficient period of time. Thus, the efficacy of antisense oligodeoxynucleotides may be improved by employing a sustained release delivery system which would protect from degradation by nucleases whilst delivering the nucleic acid in a controlled manner to the site of action. Biodegradable polymer films and micro spheres were evaluated as delivery devices for the oligodeoxynucleotides and ribozymes. Polymers such as polylactide, polyglycolide, polyhydroxybutyrate and polyhydroxyvalerate were used due to their biocompatability and non toxic degradation products. Release profiles of antisense nucleic acids from films over 28 days was biphasic, characterised by an initial burst release during the first 48 hours followed by a more sustained release. Release from films of longer antisense nucleic acids was slower compared to shorter nucleic acids. Backbone type also affected release, although to a lesser extent than length. Total release of the nucleic acids is dependent upon polymer degradation, no degradation of the polymer films was evident over the 28 day period, due to the high molecular weight and crystallinity of the polymers required to make solvent cast films. Backbone length and type did not affect release from microspheres, release was generally faster than from films, due to the increased surface area, and low molecular weight polymers which showed signs of degradation over the release period, resulting in a triphasic release profile. An increase in release was observed when sphere size and polymer molecular weight were decreased. The polymer entrapped phosphodiester oligodeoxynucleotides and ribozymes had enhanced stability compared to free oligodeoxynucleotides and ribozymes when incubated in serum. The released nucleic acids were still capable of hybridising to their target sequence, indicating that the fabrication processes did not adversely effect the properties of the antisense nucleic acids. Oligodeoxynucleotides loaded in 2μm spheres had a 10 fold increase in macrophage association compared to free oligodeoxynucleotides. Fluorescent microscopy indicates that the polymer entrapped oligodeoxynucleotide is concentrated inside the cell, whereas free oligodeoxynucleotides are concentrated at the cell membrane. Biodegradable polymers can reduce the limitations of antisense therapy and thus offer a potential therapeutic advantage.

Melt processable biomaterials for degradable surgical fixation devices

There are currently few biomaterials which combine controlled degradation rates with ease of melt processability. There are however, many applications ranging from surgical fixation devices to drug delivery systems which require such combination properties. The work in this thesis is an attempt to increase the availability of such materials. Polyhydroxybutyrate-polyhydroxyvalerate copolymers are a new class of potentially biodegradable materials, although little quantitative data relating to their in vitro and in vivo degradation behaviour exists. The hydrolytic degradation of these copolymers has been examined in vitro under conditions ranging from `physiological' to extremes of pH and elevated temperature. Progress of the degradation process was monitored by weight loss and water uptake measurement, x-ray diffractometry, optical and electron microscopy, together with changes in molecular weight by gel permeation chromatography. The extent to which the degradation mechanism could be modified by forming blends with polysaccharides and polycaprolactone was also investigated. Influence of the valerate content, molecular weight, crystallinity, together with the physical form of the sample, the pH and the temperature of the aqueous medium on the hydrolytic degradation was investigated. Its progress was characterised by an initial increase in the wet weight, with concurrent decrease in the dry weight as the amorphous regions of the polymer are eroded, thereby producing an increase in matrix porosity. With the polysaccharide blends, this initial rate is dramatically affected, and erosion of the polysaccharide from the matrix markedly increases the internal porosity which leads to the eventual collapse of the matrix, a process which occurs, but less rapidly, in the degradation of the unblended polyhydroxybutyrate-polyhydroxyvalerate copolymers. Surface energy measurement and goniophotometry proved potentially useful in monitoring the early stages of the degradation, where surface rather than bulk processes predominate and are characterised by little weight loss.