Gene delivery can potentially treat acquired and genetic diseases such as cystic fibrosis, haemophilia and cancer. Non-viral gene delivery vectors are attractive candidates over viral vectors such as recombinant viruses, due to their lower cytotoxicity and immunogenicity, despite significantly lower transfection efficiencies. To improve efficiency of non-viral vectors, the investigation of the various parameters influencing DNA transfection is essential. The present study developed a versatile gene delivery system with tailored physicochemical and biological properties. The system used polymer brushes synthesised via atomic transfer radical polymerisation (ATRP), grafted from silica nanoparticles, whose charge density, grafting density, chemistry, length of brush, the size and shape can be altered. The primary focus of the study was poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), known for its positive charge and DNA condensation. The ability of PDMAEMA to interact with DNA was characterised using dynamic light scattering, electrophoretic light scattering methods, surface plasmon resonance and in situ ellipsometry whilst its interaction with cells was studied via cell viability assays. The brush behaviour in response to pH and ionic strength was also studied. The charge density was altered by copolymerising with poly[oligo(ethylene glycol) methyl ether methacrylate](POEGMA) and the effect of such modification on DNA interaction was studied. PDMAEMA-grafted nanoparticles gave the highest transfection efficiency compared to other synthesised polymer brushes, but still displaying almost 2-fold lower transfection efficiency than the commercially available reagent jetPEI®. Different brush chemistries were also investigated. Poly(glycidyl methacrylate) (PGMA) decorated with oligoamines: allylamine, diethylenetriamine and pentaethylene hexamine, and PDMAEMA quaternized with alkyl halides: methyl iodide, allyl iodide and ethyl iodoacetate did not show any significant transfection, despite their performance reported in the literature. The robust system developed is a promising platform for further investigation of parameters influencing cellular uptake and gene expression, and important milestone to develop non-viral gene delivery systems.

Les travaux de cette thèse portent sur la formation et la polymérisation d'émulsions de Pickering stabilisées par des nanocristaux de cellulose (NCC). Tout d’abord, les NCC sont fonctionnalisés en surface de façon à modifier d'une part, leur balance hydrophile/hydrophobe et d'autre part, apporter des fonctions promoteurs de la polymérisation par ATRP de la phase interne ou externe. A l'aide de ces NCC, des émulsions directes, inverses et doubles de styrène et de monomères acryliques ont été stabilisées puis polymérisées. L'impact, de la fonctionnalisation des NCC, de la nature du monomère et de l’amorceur, de la présence ou pas de sel sur, la taille, la stabilité, la couverture des gouttes et la morphologie des objets obtenus, a été étudiée. Les latex issus de la polymérisation radicalaire d’émulsions directes de Pickering ont permis la préparation de composites aux propriétés mécaniques améliorées par rapport à celles de la matrice sans charge. Les émulsions stabilisées par des NCC réactifs ont conduit à la synthèse de capsules ou de billes pleines en fonctions de la nature du monomère polymérisé. Des matériaux poreux ont été obtenus par polymérisation des émulsions inverses de Pickering. Enfin, la polymérisation des émulsions doubles a permis l’obtention d’objets à morphologie tout à fait inédite avec l’encapsulation de capsules de polystyrène dans des capsules plus volumineuses de ce même polymère. / Pickering emulsions are based on amphiphilic particle stabilizers, which adsorb irreversibly at the liquid-liquid interface and form a rigid structure around the droplets. Amongst these particles, biosourced and biorenewable cellulose nanocrystals (CNCs) have demonstrated good performances as Pickering stabilizers for oil in water emulsions. In this thesis, a wide range of emulsions of monomers were stabilized by amphiphilic modified CNCs. These Pickering emulsions subsequently serve as vessel to perform radical polymerization. In a first step, the CNCs are modified to tailor the hydrophobic/hydrophilic balance and are used to efficiently stabilize direct (O/W), inverted (W/O) or double W/O/W Pickering emulsions of monomers. The different emulsions obtained were subsequently polymerized, by thermal radical polymerization or by SI-ATRP. The polymerization of the direct emulsions allowed producing either capsules or filled beads, depending on the monomer used, which we assigned to differences in monomer reactivity. Hence, the method offers the opportunity to tune the morphology of the polymerized spheres (empty or filled), by simply controlling the monomer conversion. The polymerization of the inverted emulsion on the other end, led to the formation of a porous material. The polymerization of the double W/O/W emulsions was also envisaged, leading original morphology such as small empty beads encapsulated into larger capsules.

Aluminium salen and aluminium salan complexes are excellent catalysts for the ring-opening polymerisation of lactide. This thesis studied their efficacy in the polymerisation of novel monomers and their ability to build new macrostructures. Aluminium salen and aluminium salan complexes were tested as catalysts for ring-opening polymerisation of common aliphatic monomers where controlled polymer synthesis has not yet been achieved with similar systems. Excellent control over molecular weight and dispersity was achieved for β-caprolactone polymerisation, with high molecular weights accessible. Immortal polymerisation could also be performed with an extremely high level of chain transfer agent (up to 100 equivalents) and the highest monomer turnover (10000 monomer equivalents) with aluminium salen catalysts to date. Addition of functional groups to the monomer was also studied; the effect of steric bulk in polymerisation of methylsubstituted derivatives was significant. Protected alcohol functionalities can also be introduced into easily synthesised homopolymers and copolymers. The first example of synthesising a polyester with aromatic functionality within the polymer backbone via polymerisation of cyclic ester monomers was studied with an aluminium salen catalyst. 2,3-Dihydro-5H-1,4-benzodioxepin-5-one polymerisation was facile and proceeded under mild conditions. The resulting polymer could be depolymerised back to starting monomer with the same aluminium salen catalyst under dilute conditions. Random, AB diblock and ABA triblock copolymers were readily synthesised with L-lactide and β-butyrolactone as comonomers. Block copolymers with β-butyrolactone could also be selectively depolymerised, to give poly(3-hydroxybutyrate) homopolymers. Attempted polymerisation of a range of other aromatic monomers was unsuccessful due to addition of steric bulk, changing orientation of the monomer ester bond or decreasing the ring size. Synthesis of homopolymer and ABA triblock copolymers with L-lactide and alkyl-substituted β-lactones was investigated. Homopolymerisation of all alkyl-substituted β-lactones resulted in well controlled polymer, with rate decreasing as alkyl-substituent length increased. A sequential addition of monomers method with β-butyrolactone, β-valerolactone and β-heptanolactone was employed for copolymer synthesis. Copolymers synthesised from β-butyrolactone and β-valerolactone resulted in tunable glass transition and melting temperatures. Copolymers synthesised from β-heptanolactone resulted in thermoplastic elastomers exhibiting microphase separation, supported by differential scanning calorimetry and small-angle X-ray scattering. Finally, optimisation of in situ generated carbonylation catalysts was studied. Optimisation of literature complexes allowed for synthesis of β-valerolactone, β- heptanolactone, β-tridecalactone, 4-chloro-β-butyrolactone and β-6-heptenolactone on relatively large scales under much easier experimental protocols. Additionally, tuning of ortho-phenylene bridged salen ligand framework gave to structure-activity relationships. Using this optimised catalyst system, 4-chloro-β-butyrolactone and β- 6-heptenolactone were prepared and used in ring opening polymerisation. Well controlled and efficient polymerisation of 4-chloro-β-butyrolactone was easily achieved with aluminium salen and salan catalysts. Homopolymers and block copolymers with poly(ethylene glycol) and β-6-heptenolactone were readily synthesised.

Polyesters have been realised as a viable replacement for slow or non-degrading petroleum derived polymers. A variety of aliphatic polyesters, e.g. poly(lactic acid), have received a lot of attention because they are produced from renewable feedstocks and have the ability to biodegrade and bioassimilate. Poly(lactic acid)'s broader family, poly(α-hydroxy acid)s, have been produced with a wide variety of properties, that has given polyesters the potential for a more diverse range of applications. However, their synthesis has proven difficult. This thesis investigates a family of 1,3-dioxolan-4-ones as a monomer source to ease difficulties in current synthetic routes. Polymerisation of the parent 1,3-dixoxolan-4-one was tested. The copolymerisation of Llactide and 1,3-dioxolan-4-one was conducted with various monomer feedstocks. Ringopening polymerisation of 1,3-dioxolan-4-one led to the formation of paraformaldehyde as a polymerisation by-product. The copolymerisation was found to be best controlled when using a coordination-insertion type catalyst. 1,3-dioxolan-4-one was also copolymerised with ε-caprolactone and β-butyrolactone to produce copolymers with various compositions. The formation of poly(lactic acid) and poly(mandelic acid) from 5-methyl-1,3-dioxolan- 4-one and 5-phenyl-1,3-dioxolan-4-one was investigated. Poly(lactic acid) and poly(mandelic acid) were synthesised with either isotactic or atactic tacticities. Molecular weights were found to be lower than the expected values. A variety of MeAl(salen) catalysts were explored for the polymerisation of 5-methyl-1,3-dioxolan-4-one and catalysts ligated with tertiary-butyl substituted salens were found to have higher rates of polymerisation and reached high conversions. Altering the diimine bridge in the ligand led to variations in rates of polymerisation and molecular weights. The cause of the decrease in molecular weight was found to be caused by a side reaction. The side reaction was bypassed by polymerising 2,2,5- trimethyl-1,3-dioxolan-4-one and 2,2-dimethyl-5-phenyl-1,3-dioxolan-4-one to form poly(lactic acid) and poly(mandelic acid), respectively, with the expulsion of acetone. The scope of 1,3-dioxolan-4-ones capable of being polymerised to form poly(α-hydroxy acid)s was expanded to include iso-propyl, cyclohexyl, normal-butyl, iso-butyl, propargyl, chloromethyl and benzyloxymethyl substituents at the five position. The glass transition temperatures accessible from this synthetic route was expanded (22-105 °C). Kinetic experiments revealed the impact of the substituents steric bulk on the rate of polymerisation and points toward a coordination-insertion mechanism. Poly(lactic acid-co-glycolic acid) was copolymerised with 5-propargyl-1,3-dioxolan-4-one to incorporate alkynyl functionality and hence Raman spectroscopy showed the polymer had a distinct peak at 2128 cm-1. Following post-polymerisation modification of poly(lactic acid-co-3-chloro-2-hydroxypropanoic acid) copolymers, acrylate functionalised polymers were produced. The copolymers were shown to be capable of crosslinking poly(α-hydroxy acid) and poly(methyl methacrylate).

Le transport intracellulaire met en jeu des vésicules et nécessite ainsi des modifications de la membrane plasmique. En particulier, des nanotubes de membrane de quelques dizaines de nanomètres peuvent se former. Nous avons mis en place un système biomimétique à base de liposomes pour décrypter les mécanismes de changement de forme membranaire, en particulier sous l’action du cytosquelette d’actine. La physique des tubes de membrane est bien connue, notamment la force nécessaire au maintien de ce type de tube, qui dépend de l’élasticité de courbure du liposome et de sa tension de membrane imposée par l’aspiration d’une micropipette. En utilisant une diode quatre quadrants, nous avons atteint une résolution temporelle de l’ordre de 4 µs, et une résolution en termes de force plus précise que le pN. Ce montage permet pour la première fois d’étudier les fluctuations de tels tubes. Cette thèse ouvre la voie à l’étude des effets de la polymérisation d’actine sur ces nanotubes / Intracellular transport involves membrane compartments and thus requires dynamic changes in the morphology of cell membranes. In this case, membrane tubes are formed whose radius is of the order of several tens of nanometers. We develop biomimetic systems based on model lipid membranes to decipher the mechanisms of membrane remodelling in particular under the action of the actin cytoskeleton. The mechanics of membrane nanotubes, especially the force needed to form and maintain a nanotube, are now well understood. The force depends on the curvature elasticity of the membrane and on its mechanical tension that is controlled in our experiment by micropipette aspiration. By using a four-quadrant diode, we obtain an unprecedented temporal resolution, in the order of 4 µs, and a force resolution under pN. This setup allows us to access unrivaled membrane nanotube properties.This thesis paves the way for studying the effect of actin dynamics on membrane nanotubes

New water soluble, attainable to ATRP polymerization Cl-terminated poly(glycidol) macroinitiators were prepared by modification of (Omega)-hydroxyl group of poly(glycidol acetal) using 2-chloropropionyl chloride fallowed by selective acidic deprotection of acetal groups. The obtained macroinitiators of different molar masses were successfully employed in ATRP of NIPAM and 4VP to give well-defined stimuli sensitive block copolymers of targeted molar ratio of blocks. The results obtained from light scattering methods showed formation of stable aggregates upon stimuli (pH or temperature) by all the obtained polymers. Additionally, photocrosslinkable block copolymers of glycidol and NIPAM having incorporated moieties of chromophore (2-(dimethyl maleinimido)-N-ethyl-acryl amide) were prepared using macroinitiator technique and used to synthesis of temperature sensitive microgels. Conjunction points have been successfully formed by UV irradiation of polymer water solution above cloud point. The influence of such parameters like block ratio, block length, amount of chromophore, concentration, irradiation time, temperature and heating rate on the properties of obtained microgels was investigated. The obtained core-shell structures were stable under critical conditions and showed continuous volume phase separation process upon increase of temperature, fully reversible and reproducible (no hysteresis effect). Thus, the proposed method not only gave the opportunity to control size or swelling degree of microgels, but also diminished gradient in crosslinking density (random chromophore distribution in polymer backbone), improved colloid stability (poly(glycidol) shell) and completely eliminated additives (surfactants, initiators, stabilizers).

This thesis is concerned with the development and characterisation of polymeric nanoporous adsorbents to be used for blood purification. Current treatment methods for suffers of chronic renal failure are limited to haemodialysis, peritoneal dialysis and organ transplant. Organ transplant is the most efficient option however lack of donor organs mean that the majority of suffers rely on dialysis. Unfortunately both dialysis treatments are lacking when it comes to the removal of middle molecular weight molecules (MMs) (500 - 60000 Da) and the accumulation of these molecules has been attributed to a number of additional ailments suffered by those on long term dialysis. Sorbent augmented dialysis has been identified as a potential avenue to remove these MMs, an additional column would be introduced to the haemodialysis loop this would contain adsorbent particles to remove these unwanted molecules. Styrene-divinylbenzne copolymers have been identified as suitable for this task as they will non-specifically adsorb a wide range of molecules. One major concern with the introduction of a polymeric adsorbent is the potential removal of human serum albumin HSA from the patient's blood, this essential blood protein is present in very high concentrations typically 40g/l and this will potentially swamp the surface of any adsorbent. Fortunately HSA is a large blood protein (69kDa) and as such the method to combat this limitation as explored in this thesis is to tailor the pore structure of the polymeric adsorbent to size exclude albumin while retaining sufficient adsorption capacity to remove the MMs. To achieve these goals a number of polymeric adsorbents were generated using different porogens and degrees of crosslinking to control the porous structure. These adsorbents were analysed using a number of characterisation methods to assess their dry and swollen state porosities and molecular weight cut offs. Once a suitable material had been developed protein adsorption studies were carried out to confirm the size exclusion of HSA and the uptake of MMs.

Miniemulsion technology is attracting increasing interest for the preparation of nano-size particles. However, the barrier to industrialising miniemulsion-based products is the lack of an energy efficient and scalable homogenisation device. Current laboratory techniques consist of batch units, however trends are leaning towards developing continuous processes. The objective of the work presented here is to investigate the use of the rotor-stator (RS) and static mixers (SM) as homogenisation devices and ultimately develop a continuous emulsification/polymerisation process for the preparation of miniemulsions. Initially we investigated the RS as a homogenisation device and found that we were able to generate droplets ranging from 300 nm to 2 μm, at industrially pertinent solids content. Subsequently, we investigated the use of SM and compared their performance in terms of mean droplet size evolutions with the rotor-stator. We were able to generate droplets < 200 nm in size and polymerise them in a stable fashion. All the available emulsification devices were then compared in terms of power/energy consumption, droplet size distributions and shear rates. It was observed that with energy costs being of similar orders of magnitude, SM imposed less shear, produced relatively narrow distributions and were better adapted to scale-up, making them the optimal choice for miniemulsification. Energy savings were increased by reducing coalescence during the emulsification step by using in situ generated surfactants, ultimately reducing emulsification time. Neutralising a water-soluble base with an oil-soluble acid almost instantaneously generates in situ surfactants at the oil-water interface. The reduction in emulsification time was partially attributed to the elimination of the relatively slow adsorption step typical of preformed surfactants. These results were used to show that emulsifying in situ formulations at moderate flow rates in line with SMX mixers can substantially reduce emulsification time from 30 minutes, with preformed surfactants, to ~30 seconds. With such a rapid emulsification step, it was possible to test the feasibility of the continuous emulsification process followed by polymerisation in a tubular reactor. Comparing this process with a batch operation, similar results for the ratio of NP/ND and conversion were obtained, but the continuous process was accomplished in a single step. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2010-04-19 03:32:08.766

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 10 uppsatser.

Freiburg i. Br., Univ., Diss., 2007.