Synthesis of polymers and copolymers of lactide through titanium initators

The research described in this thesis explored the synthesis tlnd characteristltion of biocompatible and biodegradable polymers of lactide through non-toxic titanium alkoxide nitiators. The research objectives focused on the preparation of polylactides in both solvent and solventless media, to produce materials with a wide range of molecular weights. The polylactides were fully characterised using gel permeation chromatography and 1H and 13C NMR spectroscopy. NMR spectroscopy was carried out in the study the reaction mechanisms. Kinetic studies of the ring opening polymerisation of lactide with titanium alkoxide initiators were also conducted using NMR spectroscopy. The objectives of this research were also focused on the enhancement of the flexibility of the polymer chains by synthesising random and block copolymers of lactide and ε-caprolactone using Ti(0-i-Pr)4 as an initiator, This work involved extensive characterisalion of the synthesised copolymers using gel permeation chromatography and 1H and 13C NMR spectroscopic analysis. Kinetic studies of the ring opening polymerisation of ε-caplrolactone and of the copolymerisation of lactide and ε-caprolactone with Ti(O-i-Pr)4 as an initiator were also carried out. The last section of this work involved the synthesis of block and star-shaped copolymers of lactide and poly(ethylene glycol) [PEG]. The preparation of lactide/PEG block copolymers was carried out by ring opening polymerisation of L-Iactide using Ti(O-i-Pr)4 as an initiator and hydroxyl-terminated PEG's with different numbers of hydroxyl groups as co-initiators both in solution and solventless media. These all-in-one polymersations yielded the synthesis of both lactide homopolymer and lactide/PEG block copolymer. In order to selectively synthesise copolymers of lactide and PEG, the experiment was carried out in two steps. The first step consisted of the synthesis of a titanium macro-initiator by exchanging the iso-propoxide ligands by PEG with different numbers of hydroxyl groups. The second step involved the ring opening polymerisation of lactide using the titanium macrocatalyst that was prepared as an initiator. The polymerisations were carried out in a solventless media. The synthesis of lactide/PEG copolymers using polyethylene glycol with amino terminal groups was also discussed. Extensive characterisation of the lactide block copolymers and macroinitiators was carried out using techniques such as, gel permeation chromatography (GPC), NMR spectroscopy and differential scanning calorimeter (DeS).

The suitability of biodegradable poly(dl-lactide-co-glycolide)75:25 microspheres for the sustained release of antibiotics

Post-operative infections resulting from total hip arthroplasty are caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa entering the wound perioperatively or by haemetogenous spread from distant loci of infection. They can endanger patient health and require expensive surgical revision procedures. Gentamicin impregnated poly (methyl methacrylate) bone cement is traditionally used for treatment but is often removed due to harbouring bacterial growth, while bacterial resistance to gentamicin is increasing. The aim of this work was to encapsulate the antibiotics vancomycin, ciprofloxacin and rifampicin within sustained release microspheres composed of the biodegradable polymer poly (dl-lactide-co-glycolide) [PLCG] 75:25. Topical administration to the wound in hydroxypropylmethylcellulose gel should achieve high local antibiotic concentrations while the two week in vivo half life of PLCG 75:25 removes the need for expensive surgical retrieval operations. Unloaded and 20% w/w antibiotic loaded PLCG 75:25 microspheres were fabricated using a Water in Oil emulsification with solvent evaporation technique. Microspheres were spherical in shape with a honeycomb-like internal matrix and showed reproducible physical properties. The kinetics of in vitro antibiotic release into newborn calf serum (NCS) and Hank's balanced salt solution (HBSS) at 37°C were measured using a radial diffusion assay. Generally, the day to day concentration of each antibiotic released into NCS over a 30 day period was in excess of that required to kill St. aureus and Ps. auruginosa. Only limited microsphere biodegradation had occurred after 30 days of in vitro incubation in NCS and HBSS at 37°C. The moderate in vitro cytotoxicity of 20% w/w antibiotic loaded microspheres to cultured 3T3-L1 cells was antibiotic induced. In conclusion, generated data indicate the potential for 20% w/w antibiotic loaded microspheres to improve the present treatment regimens for infections occurring after total hip arthroplasty such that future work should focus on gaining industrial collaboration for commercial exploitation.

Poly (D,L-Lactide) based microspheres for pulmonary delivery of proteins

Initial work focused on the preparation, optimisation and characterisation of poly (D,L-lactide) (PLA) microspheres with the aim of optimising their formulation based on minimizing the particle size into the range suitable for pulmonary delivery to alveoli. In order to produce dry powders and to enhance their long-term physico-chemical stability, microspheres were prepared as a dry powder via freeze-drying. Optimisation studies showed that using appropriate concentrations of polymer 3% (w/v) in organic phase and emulsifier 10% (w/v) in external aqueous phase, the double solvent evaporation method produced high protein loading microspheres (72 ± 0.5%) with an appropriate particle size for pulmonary drug delivery. Combined use of trehalose and leucine as cyroprotectants (6% and 1% respectively, w/v) produced freeze-dried powders with the best aerosolisation profile among those tested. Although the freeze-dried PLA microsphere powders were not particularly respirable in dry powder inhalation, nebulisation of the rehydrated powders using an ultrasonic nebuliser resulted in improved aerosilisation performance compared to the air-jet nebuliser. When tested in vitro using a macrophage cell line, the PLA microspheres system exhibited a low cytotoxicity and the microspheres induced phagocytic activity in macrophages. However, interestingly, the addition of an immunomodulator to the microsphere formulations (4%, w/w of polymer) reduced this phagocytic activity and macrophage activation compared to microspheres formulated using PLA alone. This suggested that the addition of trehalose dibehenate may not enhance the ability of these microspheres to be used as vaccine delivery systems.

Novel three-component blends based on poly(L-lactide)

A significant number of poly a-ester homologues of poly(L-lactide) (PLLA) have been synthesized and used in miscibility studies together with conventional isomeric diacid-diol polyester variants, poly ß-esters (based on ß-hydroxybutyrate (HB) and ß-hydroxyvalerate (HV)), poly e-caprolactone (PCL), poly e-caprolactone copolymers (e.g. poly(L-lactide-co-caprolactone), and a series of cellulose-based polymers (e.g. cellulose acetate butyrate (CAB), cellulose acetate propionate (CAP)). A combinatorial approach to rapid miscibility screening using 96-well plates and a uv-visible multi-wavelength plate reader has been developed enabling the clarity of PLLA-based multi-component blend films to be observed. Using these techniques and materials, the ternary phase compatibility diagrams of a range of three-component blend films was prepared, illustrating ranges of behavior varying from miscible blends giving rise to clear films to immiscible blends which are opaque. In this way, novel three-component blends of PLLA/CAB/PCL were developed which are miscible when the CAB content is more than 30%, PLLA less than 80% and PCL less than 60%.

Multifunctional bioactive glass scaffolds coated with layers of poly(d,l-lactide-co-glycolide) and poly(n-isopropylacrylamide-co-acrylic acid) microgels loaded with vancomycin

A new family of multifunctional scaffolds, incorporating selected biopolymer coatings on basic Bioglass® derived foams has been developed. The polymer coatings were investigated as carrier of vancomycin which is a suitable drug to impart antibiotic function to the scaffolds. It has been proved that coating with PLGA (poly(lactic-co-glycolic acid)) with dispersed vancomycin-loaded microgels provides a rapid delivery of drug to give antibacterial effects at the wound site and a further sustained release to aid mid to long-term healing. Furthermore, the microgels also improved the bioactivity of the scaffolds by acting as nucleation sites for the formation of HA crystals in simulated body fluid. © 2013 Elsevier B.V. All rights reserved.

Controlled synthesis and processing of a poly(L-lactide-co-ε-caprolactone) copolymer for biomedical use as an absorbable monofilament surgical suture

Poly(L-lactide-co-ε-caprolactone) 75:25% mol, P(LL-co-CL), was synthesized via bulk ring-opening polymerisation (ROP) using a novel tin(II)alkoxide initiator, [Sn(Oct)]2DEG, at 130oC for 48 hrs. The effectiveness of this initiator was compared withthe well-known conventional tin(II) octoateinitiator, Sn(Oct)2. The P(LL-co-CL) copolymersobtained were characterized using a combination of analytical technique including: nuclear magnetic resonance spectroscopy (NMR), differential scanning calorimetry (DSC), thermogravimetry (TG) and gel permeation chromatography (GPC). The P(LL-co-CL) was melt-spun into monofilament fibres of uniform diameter and smooth surface appearance. Modification of the matrix morphology was then built into the as-spun fibresvia a series of controlled off-line annealing and hot-drawing steps. © (2014) Trans Tech Publications, Switzerland.

Preparation and property testing of compatibilized poly(l-lactide)/thermoplastic polyurethane blends

Poly(l-lactide) (PLL) has been blended with a polycaprolactone-based thermoplastic polyurethane (TPU) elastomer as a toughening agent and a poly(l-lactide-co-caprolactone) (PLLCL) copolymer as a compatibilizer. Both 2-component (PLL/TPU) and 3-component (PLL/TPU/PLLCL) blends were prepared by melt mixing, characterized, hot-pressed into thin sheets and their tensile properties tested. The results showed that, although the TPU could toughen the PLL, the blends were largely immiscible leading to phase separation. However, addition of the PLLCL copolymer improved blend compatibility. The best all-round properties were found for the 3-component blend of composition PLL/TPU/PLLCL = 90/10/10 parts by weight.

Preparation of a poly(L-lactide-co-caprolactone) copolymer using a novel tin(II) alkoxide initiator and its fiber processing for potential use as an absorbable monofilament surgical suture

A poly(L-lactide-co-caprolactone) copolymer, P(LL-co-CL), of composition 75:25 mol% was synthesized via the bulk ring-opening copolymerization of L-lactide and ε-caprolactone using a novel bis[tin(II) monooctoate] diethylene glycol coordination-insertion initiator, OctSn-OCH2CH2OCH2CH2O-SnOct. The P(LL-co-CL) copolymer obtained was characterized by a combination of analytical techniques, namely nuclear magnetic resonance spectroscopy, gel permeation chromatography, dilute-solution viscometry, differential scanning calorimetry, and thermogravimetric analysis. For processing into a monofilament fiber, the copolymer was melt spun with minimal draw to give a largely amorphous and unoriented as-spun fiber. The fiber's oriented semicrystalline morphology, necessary to give the required balance of mechanical properties, was then developed via a sequence of controlled offline hot-drawing and annealing steps. Depending on the final draw ratio, the fibers obtained had tensile strengths in the region of 200–400 MPa.

PLGA microspheres for the delivery of a novel subunit TB vaccine

Biodegradable poly(dl-lactide-co-glycolide) microspheres were prepared using a modified double emulsion solvent evaporation method for the delivery of the subunit tuberculosis vaccine (Ag85B-ESAT-6), a fusion protein of the immunodominant antigens 6-kDa early secretory antigenic target (ESAT-6) and antigen 85B (Ag85B). Addition of the cationic lipid dimethyl dioctadecylammonium bromide (DDA) and the immunostimulatory trehalose 6,6'-dibehenate (TDB), either separately or in combination, was investigated for the effect on particle size and distribution, antigen entrapment efficiency, in vitro release profiles and in vivo performance. Optimised formulation parameters yielded microspheres within the desired sub-10 mu m range (1.50 +/- 0.13 mu m), whilst exhibiting a high antigen entrapment efficiency (95 +/- 1.2%) and prolonged release profiles. Although the microsphere formulations induced a cell-mediated immune response and raised specific antibodies after immunisation, this was inferior to the levels achieved with liposomes composed of the same adjuvants (DDA-TDB), demonstrating that liposomes are more effective vaccine delivery systems compared with microspheres.

Design and characterisation of novel blends of poly(lactic acid)

This thesis is concerned with the effect of polymer structure on miscibility of the three component blends based on poly(lactic acid) (PLA) with using blending techniques. The examination of novel PLA homologues (pre-synthesised poly(a-esters)), including a range of aliphatic and aromatic poly(a-esters) is an important aspect of the work. Because of their structural simplicity and similarity to PLA, they provide an ideal system to study the effect of polyester structures on the miscibility of PLA polymer blends. The miscibility behaviour of the PLA homologues is compared with other aliphatic polyesters (e.g. poly(e-caprolactone) (PCL), poly(hydroxybutyrate hydroxyvalerate) (P(HB-HV)), together with a series of cellulose-based polymers (e.g. cellulose acetate butyrate (CAB)). The work started with the exploration the technique used for preliminary observation of the miscibility of blends referred to as “a rapid screening method” and then the miscibility of binary blends was observed and characterised by percent transmittance together with the Coleman and Painter miscibility approach. However, it was observed that symmetrical structures (e.g. a1(dimethyl), a2(diethyl)) promote the well-packing which restrict their chains from intermingling into poly(L-lactide) (PLLA) chains and leads the blends to be immiscible, whereas, asymmetrical structures (e.g. a4(cyclohexyl)) behave to the contrary. a6(chloromethyl-methyl) should interact well with PLLA because of the polar group of chloride to form interactions, but it does not. It is difficult to disrupt the helical structure of PLLA. PLA were immiscible with PCL, P(HB-HV), or compatibiliser (e.g. G40, LLA-co-PCL), but miscible with CAB which is a hydrogen-bonded polymer. However, these binary blends provided a useful indication for the exploration the novel three component blends. In summary, the miscibility of the three-component blends are miscible even if only two polymers are miscible. This is the benefit for doing the three components blend in this thesis, which is not an attempt to produce a theoretical explanation for the miscibility of three components blend system.

Degradable poly(ethylene glycol)-based hydrogels:synthesis, physico-chemical properties and in vitro characterization

Designing degradable hydrogels is complicated by the structural and temporal complexities of the gel and evolving tissue. A major challenge is to create scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. Poly(ethylene glycol) was used in this work, to form biodegradable poly(ethylene glycol)-based hydrogels with hydrolyzable poly-l-lactide segments in the backbone. Non-degradable poly(ethylene glycol) was also introduced in the formulation to obtain control of the degradation profile that encompasses cell growth and new tissue formation. The dependence on polymer composition was observed by higher degradation profiles and decreased mechanical properties as the content of degradable segments was increased in the formulation. Based on in vitro tests, no toxicity of extracts or biomaterial in direct contact with human adipose tissue stem cells was observed, and the ultraviolet light treatment did not affect the proliferation capacity of the cells.

Liposomes act as stronger sub-unit vaccine adjuvants when compared to microspheres

The ability of liposomes and microspheres to enhance the efficacy of a sub-unit antigen was investigated. Microspheres were optimised by testing a range of surfactants employed in the external aqueous phase of a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation process for the preparation of microspherescomposed of poly(d,l-lactide-co-glycolide) and the immunological adjuvant dimethyl dioctadecyl ammonium bromide (DDA)and then investigated with regard to the physico-chemical and immunological characteristics of the particles produced. The results demonstrate that this parameter can affect the physico-chemical characteristics of these systems and subsequently, has a substantial bearing on the level of immune response achieved, both humoural and cell mediated, when employed for the delivery of the sub-unit tuberculosis vaccine antigen Ag85B-ESAT-6. Moreover, the microsphere preparations investigated failed to initiate immune responses at the levels achieved with an adjuvant DDA-based liposome formulation (DDA-TDB), further substantiating the superior ability of liposomes as vaccine delivery systems.

Administration routes affect the quality of immune responses:a cross-sectional evaluation of particulate antigen-delivery systems

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.

Synthesis and evaluation of novel polymer modifiers for biodegradable polymers

The effects of ester plasticizers and copolymers on the mechanical properties of the natural biodegradable polymers, poly(3-hydroxybutyrate) [PHB] and poly(lactic acid) [PLA] have been studied after subjecting to melt processing conditions. Ester plasticizers were synthesized from citric, tartaric and maleic acids using various alcohols. A variety of PLA copolymers have also been prepared from poly(ethylene glycol) derivatives using stannous octanoate catalysed ring opening polymerisations of DL-lactide. A novel PLA star copolymer was also prepared from an ethoxylated pentaerythritol. The structures of these copolymers were determined by NMR spectroscopy. The plasticizing effect of the synthesised additives at various concentrations was determined. While certain additives were capable of improving the mechanical properties of PLA, none were effective in PHB. Moreover, it was found that certain combinations of additives exhibited synergistic effects. Possible mechanisms are discussed. Biotic and abiotic degradation studies showed that the plasticizers (esters and copolymers) did not inhibit the biodegradability of PHB or PLA in compost at 60°C. Simple toxicity tests carried out on compost extract and its ability to support the growth of cress seeds was established. PLA was found to be susceptible to limited thermal degradation under melt processing conditions. Conventional phenolic antioxidants showed no significant effect on this process, suggesting that degradation was not predominantly a free radical process. PLA also underwent photo-oxidative degradation with UV light and the process could be accelerated in the presence of a photoactivator such as iron (III) diisononyl dithiocarbamate. The mechanisms for the above processes are discussed. Finally, selected compounds were prepared on a pilot plant scale. Extruded and blown films were prepared containing these additives with conventional polymer processing equipment. The mechanical properties were similar to those obtained with laboratory produced compression moulded films.

Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).