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The Enhanced Permeability and Retention (EPR) effect has seen considerable exploration by many researchers since it’s discovery by Maeda et al in 1985. Polymers and nanoparticles with a long blood residence half-life can accumulate in some tumour tissues, allowing for the delivery of either diagnostic or therapeutic payloads. We have contributed to this field by the development of methodology to prepare radiolabeled dendrimers which are suitable for EPR effect accumulation with a variety of peripheral functionalities. The first of these was a 99mTc-labeled fifth generation dendron which was peripherally functionalized with low molecular weight poly(ethylene glycol) chains, which was observed to accumulate in xenograft mouse tumours over the course of 6 hours. This work led to the development of improved synthetic means for the preparation of high generation dendrimers with complex peripheral functionality, which hinged on the use of the Strain Promoted Alkyne-Azide Cycloaddition reaction to give high generation dendrimers by a convergent approach. This resulted in the facile preparation of dendrimers with challenging peripheral functionality in reaction times as short as 5 minutes. This SPAAC based convergent synthesis approach was used to prepare 99mTc labeled sulfobetaine and carboxybetaine dendrons of the sixth generation, and these compounds were found to have a size greater than the renal clearance threshold of ~ 5 nm, though it was found that labeling with [99mTc(CO)3]+ was not possible without extensive degradation of the zwitterionic dendrimers. Finally, the dendritic architecture explored for imaging was adapted for use in shielding an enzyme from macromolecules while retaining activity against the native small molecule substrate, and we found that conjugation of high-generation bis-MPA dendrons to α-chymotrypsin was an effective way to eliminate enzyme activity against macromolecules while preserving efficacy against small substrates, indicating this approach may be an effective way to shield proteins from the immune system without interfering with their desired function. This work illustrates the ability to radiolabel polyester dendrimers for tumour imaging through the EPR effect. In addition, it has demonstrated that polymer architecture has a large impact on the properties of polymer-protein conjugates and gives evidence of unique properties that are imparted by the conjugation of high-generation dendrimers onto a protein. / Thesis / Doctor of Science (PhD)

Cette étude porte sur le développement de nanoparticules pour différentes applicationsbiologiques. Trois systèmes de nanoparticules ont été mis au point : des clusters de nanoparticulesmagnétiques pour l’extraction par magnétophorèse d’objets biologiques, des agents de contrastemultimodaux (IRM, fluorescence dans le proche infrarouge) pour le diagnostic de l’athérosclérose etdes nanoparticules de silice fluorescentes doublement marquées pour la détection de tumeurs in vivo.Au cours de cette étude, une stratégie de greffage de surface de silice par des macromolécules depoly(oxyde d’éthylène) (PEG) permettant d’atteindre de hautes densités de greffage. Cette PEGylationpermet, d’annuler les interactions non spécifiques dans le cadre de l’extraction magnétique rendantainsi ce système plus efficace, et de conférer aux nanoparticules des propriétés de furtivité vis-à-vis dusystème immunitaire dans le cadre du marquage de tumeurs. Le contrôle du nombre de biomoléculesgreffées régiosélectivement sur les nanoparticules (Annexine A5, ou fragments d’anticorps) ainsi quel’étude des interactions biomoléculaires par des techniques de biophysique (SPR, QCM-D) ont permisd’optimiser la propriété de reconnaissance des nano-objets pour leurs cible respective. Enfin, les nanoobjetsont été évalués dans le cadre de leur application. / The aim of this study was the design of nanoparticles for three different biologicalapplications: magnetic nanoparticles cluster for magnetic extraction of biological materials,multimodal contrast agents (MRI and near infrared fluorescence imaging) for atherosclerosisdiagnosis, and fluorescent silica nanoparticles with two different dyes for in vitro and in vivo tumorlabeling. One part of the project dealt with the developement of a new grafting method ofpoly(ethylene oxide) macromolecules onto nanoparticle’s silica surfaces (PEGylation) in order toobtain a high grafting densities. The obtained results have shown that this PEGylation reduces the nonspecificprotein adsorption allowing a better extraction and sorting efficiency, and also permitsnanoparticles to escape the surveillance of the immune system for in vivo tumor labeling. Therefore,the biomolecular recognition of the nanoparticles has been optimized by controlling the number ofconjugated biomolecules and by studying this biomolecular recognition using biophysical methods(SPR, QCM-D). Finally, the different nano-objects were evaluated in the context of their respectiveapplication.

Dans ce travail, les copolymérisations radicalaires contrôlées de monomères donneurs (dérivés du styrène) et accepteurs (maleimides N-substitués) ont été effectuées afin de préparer des polymères à séquences contrôlées. Ces macromolécules ont été préparées par polymérisation radicalaire contrôlée par la voie des nitroxides en utilisant le SG1 comme agent de contrôle. Des polymères ayant des microstructures bien définies ont été obtenus par le contrôle du temps de l’addition d’une petite quantité de monomère accepteur au cours de la polymérisation d’un large excès de monomère de type donneur. Dans cette thèse, des nouveaux dérivés styréniques para-substitués ont été sélectionnés afin de préparer une variété de polymères fonctionnels à séquences contrôlées. Par exemple, des polyélectrolytes à base de poly(4-hydroxystyrène)s et poly(vinyl benzyle amine)s ont été obtenus par polymérisation de dérivés protégés du styrène (4-tert-butoxystyrène, 4-acetoxystyrène et N-(p-vinyl benzyl)phthalimide) avec une quantité non-stœchiométrique de maleimides N-substitués. Par ailleurs, des polymères PEGylés biocompatibles et solubles dans l’eau ont également été étudiés. Des polymères à séquences contrôlées portant des fonctions alcynes protégées sur chaque unité de styrène ont été dans un premier temps synthétisés. La suppression de ces groupes protecteurs a permis le greffage du α-méthoxy-ω-azido-PEG sur les fonctions alcynes libres en employant la chimie click de type CuAAC. Finalement, des polymères semi-cristallins à séquences contrôlées ont été élaborés en utilisant le styrène d’octadécyle comme monomère donneur. Les propriétés thermiques de ces polymères ont été étudiées afin d’évaluer l’influence de la microstructure sur le comportement de leur cristallisation. / In this work, controlled radical copolymerizations of donor (styrenic derivatives) and acceptor monomers (N-substituted maleimides, MIs) have been investigated in order to synthesize sequence-controlled polymers. These macromolecules were prepared by nitroxide mediated polymerization using the nitroxide SG1 as a control agent. Polymers with defined microstructures were obtained by time-controlled addition of small amounts of acceptor monomers during the polymerization of a large excess of donor monomer. In this thesis, new styrenic derivatives have been studied in order to design sequence-controlled polymers with functional backbones. For example, sequence-controlled polyelectrolytes based on poly(4-hydroxystyrene)s and poly(vinyl benzyl amine)s were obtained through the polymerization of protected styrenic derivatives (i.e. 4-tert-butoxystyrene, 4-acetoxystyrene and N-(p-vinyl benzyl)phthalimide) with non-stoichiometric quantities of N-substituted maleimides. Furthermore, the preparation of PEGylated biocompatible water-soluble polymers was also investigated. Sequence-controlled polymers bearing protected alkyne functional groups on each styrene units were first synthesized followed by the removal of their protecting groups allowing the grafting of α-methoxy-ω-azido-PEG on free alkyne moieties via CuAAC mediated click reaction. Finally, sequence-controlled semi-crystalline polymers were synthesized using octadecylstyrene as a donor monomer. The thermal properties of these polymers were studied to evaluate the influence of polymer microstructure on crystallization behavior.

Modélisations moléculaires réalisés avec le logiciel HyperChem 8. / L’effet de différentes alkylations sur l’activité biologique et la stabilité enzymatique d’un peptide linéaire L, énantiomère du modulateur allostérique des récepteurs prostaglandine F2α ont été étudiés. Dans une étude antérieure, le peptide D PDC-31 avait montré un potentiel d’inhibition des contractions du myomètre et permettait de retarder l’accouchement dans des modèles animaux et humains. Il est possible que le peptide L possède une activité semblable, mais les protéases, abondantes dans le tissu myométrial, le dégradent probablement avant qu’il ne puisse atteindre le site actif. La synthèse peptidique sur support solide suivie d’une amination réductive a permis d’obtenir différents peptides portant différentes chaines alkyle et PEG N-terminales. La protection de l’amine terminale par un groupement ortho-nitrobenzène sulfonyle suivie par une réaction de Mitsunobu a permis l’obtention d’un analogue portant une chaine farnesyle. Malgré le fait que ni l’analogue PEGylé, ni l’analogue farnesylé n’aient montrés la moindre activité, certains analogues alkylés se sont avérés actifs dans l’essai tissulaire de contractions myométriales. Le peptide L portant une chaine dodecyle s’est avéré posséder une activité statistiquement significative et reproductible. Qui plus est, l’analogue D du peptide possédant une chaine de 12 carbones s’est avéré posséder une activité inférieure à l’analogue L portant la même chaine, ce qui représente une perte d’activité significative par rapport au peptide D nonmodifié (PDC-31). / The application of hydrophobic grafts to prolong the biological activity of rapidly metabolized peptides has been explored by modification of the L-peptide of the prostaglandin F2α receptor modulator PDC-31. The all-D peptide PDC-31 has previously been shown to inhibit myometrial contractions and delay labour in various animal models as well as in humans. The L-peptide may have activity; however, proteases, which are abundant in myometrial tissue, may likely degrade the peptide before it is capable of showing activity. Solid-phase peptide synthesis followed by Nterminal modification by reductive aminations with different aldehydes provided linear aliphatic alkyl and PEG-grafted peptide analogs. Alternatively, ortho-nitrobenzensulfonylation of the peptide followed by Mitsunobu alkylation with farnesol and deprotection gave a farnesylated analog. Although the PEG and fanesylated analogs exhibited no activity, certain N-alkyl analogs exhibited inhibitory activity on myometrial contractions, with the most active analog possessing a dodecyl chain. Moreover, the N-dodecyl analog of PDC-31, exhibited lower activity than its L-counterpart in the myometrial contraction assay, and with reduced potency relative to its unmodified structure.

A enzima L-asparaginase (ASNase) de Escherichia coli é amplamente utilizada para tratar a leucemia linfoblástica aguda (LLA). No entanto, a enzima nativa pode ativar o sistema imune do hospedeiro causando reações alérgicas. A forma peguilada da enzima (PEG-ASNase) não só reduz o efeito imunológico, mas também possui a vantagem de aumentar sua meia-vida plasmática devido à menor degradação por proteases. Não obstante, a conjugação de PEG à ASNase é de forma aleatória e resulta em elevado grau de polidispersão. Nesse trabalho, um protocolo de peguilação N-terminal sítio-específica para a enzima ASNase foi desenvolvido, empregando-se metoxi-polietilenoglicol carboximetil N-hidroxisuccinimidil ester (mPEG-NHS, MW = 10 kDa). Os parâmetros de reação investigados foram a força iônica do tampão, o pH e o tempo de reação para otimizar o rendimento e minimizar a polidispersão. Os resultados confirmaram a ocorrência da reação de peguilação e baixa polidispersão. A força iônica do tampão favoreceu a conjugação N-terminal em 100 mM de tampão fosfato salino (PBS). Também observamos que o aumento do pH e tempo de reação estão diretamente relacionados com o aumento de formas polipeguiladas. O maior rendimento de monoPEG-ASNase, 42%, correspondeu à condição de reação de pH 7,5, tempo de reação de 30 min e razão PEG:enzima de 25:1. A monoPEG-ASNase foi purificada em coluna aniônica forte com gradiente linear de NaCl até 170 mM e a fração monopeguilada foi eluída em NaCl 78 mM com grau de pureza de 70% (rendimento de 55%). A cromatografia de exclusão por tamanho aumentou a pureza da monoPEG-ASNase para 99% (rendimento final de 45%). A monoPEG-ASNase, 190 ± 10 kDa, manteve sua atividade específica por 21 dias à 4ºC, enquanto que a enzima não peguilada (controle), 146 ± 6 kDa, teve uma queda de aproximadamente 52% na atividade específica em 7 dias à 4ºC. Um estudo de cinética enzimática foi realizado e obtivemos valores de kM semelhantes para as formas da enzima nativa e monopeguilada, 20 µM e 35 µM respectivamente. A monoPEG-ASNase apresentou-se mais resistente às proteases plasmáticas asparagina endopeptidase e catepsina B, por 84 h à 37 °C e apresentou ser citotóxica para células leucêmicas (MOLT-4 e Reh). Portanto, a peguilação N-terminal sítio-específica resultou em uma nova variante da ASNase que reteve grande parte de seu poder catalítico e pode ser considerada promissora para o emprego no tratamento da LLA. / The enzyme L-asparaginase (ASNase) from Escherichia coli is widely used to treat acute lymphoblastic leukemia (LLA). However, it can activate the host causing allergic reactions. Hence, the pegylated form of the enzyme (PEG-ASNase) not only reduces the immune effect but also increases plasma half-life. Nonetheless, the available PEG-ASNase is randomly pegylated and, consequently, with high degree of polydispersity. In this work we developed a site-specific N-terminus pegylation protocol for ASNase using methoxy-polyethylene glycol carboxymethyl N-hydroxysuccinimidyl ester (mPEG-NHS, MW = 10 kDa). Reaction parameters investigated were ionic strength, pH and reaction time to optimize yield and minimize polydispersity. Results confirmed pegylation and low polydispersity. Buffer ionic strength favored N-terminal conjugation at 100 mM phosphate buffer saline (PBS). Additionally, pH and reaction time were found to increase polyPEGylated species. The highest yield of monoPEG-ASNase, 42%, corresponded to reaction conditions of pH 7.5, reaction time of 30 min and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was purified in a strong anionic column with NaCl linear gradient up to 170 mM and monoPEG-ASNase was eluted with 78 mM NaCl, 70% pure (55% yield). Size exclusion chromatography was further performed and increased monoPEG-ASNase purity to 99% (45% final yield). The monoPEG-ASNase, 190 ± 10 kDa was stable for 21 days at 4ºC while nonpegylated ASNase (control), 146 ± 6 kDa, lost 52% of its activity within 7 days at 4ºC. Enzyme kinetics were studied and similar kM values were obtained for both native and monoPEG-ASNase, 20 µM and 35 µM respectively. The monoPEG-ASNase demonstrated to be resistant to the plasma proteases asparaginyl endopeptidase and cathepsin B, 84 h at 37 °C and also was cytotoxic to leukemic cells (MOLT-4 and Reh). Therefore, site-specific N-terminus pegylation of ASNase resulted in a novel enzyme variant with preserved catalytic activity and, therefore, promising for the treatment of LLA.

A enzima L-asparaginase (ASNase) de Escherichia coli é amplamente utilizada para tratar a leucemia linfoblástica aguda (LLA). No entanto, a enzima nativa pode ativar o sistema imune do hospedeiro causando reações alérgicas. A forma peguilada da enzima (PEG-ASNase) não só reduz o efeito imunológico, mas também possui a vantagem de aumentar sua meia-vida plasmática devido à menor degradação por proteases. Não obstante, a conjugação de PEG à ASNase é de forma aleatória e resulta em elevado grau de polidispersão. Nesse trabalho, um protocolo de peguilação N-terminal sítio-específica para a enzima ASNase foi desenvolvido, empregando-se metoxi-polietilenoglicol carboximetil N-hidroxisuccinimidil ester (mPEG-NHS, MW = 10 kDa). Os parâmetros de reação investigados foram a força iônica do tampão, o pH e o tempo de reação para otimizar o rendimento e minimizar a polidispersão. Os resultados confirmaram a ocorrência da reação de peguilação e baixa polidispersão. A força iônica do tampão favoreceu a conjugação N-terminal em 100 mM de tampão fosfato salino (PBS). Também observamos que o aumento do pH e tempo de reação estão diretamente relacionados com o aumento de formas polipeguiladas. O maior rendimento de monoPEG-ASNase, 42%, correspondeu à condição de reação de pH 7,5, tempo de reação de 30 min e razão PEG:enzima de 25:1. A monoPEG-ASNase foi purificada em coluna aniônica forte com gradiente linear de NaCl até 170 mM e a fração monopeguilada foi eluída em NaCl 78 mM com grau de pureza de 70% (rendimento de 55%). A cromatografia de exclusão por tamanho aumentou a pureza da monoPEG-ASNase para 99% (rendimento final de 45%). A monoPEG-ASNase, 190 ± 10 kDa, manteve sua atividade específica por 21 dias à 4ºC, enquanto que a enzima não peguilada (controle), 146 ± 6 kDa, teve uma queda de aproximadamente 52% na atividade específica em 7 dias à 4ºC. Um estudo de cinética enzimática foi realizado e obtivemos valores de kM semelhantes para as formas da enzima nativa e monopeguilada, 20 µM e 35 µM respectivamente. A monoPEG-ASNase apresentou-se mais resistente às proteases plasmáticas asparagina endopeptidase e catepsina B, por 84 h à 37 °C e apresentou ser citotóxica para células leucêmicas (MOLT-4 e Reh). Portanto, a peguilação N-terminal sítio-específica resultou em uma nova variante da ASNase que reteve grande parte de seu poder catalítico e pode ser considerada promissora para o emprego no tratamento da LLA. / The enzyme L-asparaginase (ASNase) from Escherichia coli is widely used to treat acute lymphoblastic leukemia (LLA). However, it can activate the host causing allergic reactions. Hence, the pegylated form of the enzyme (PEG-ASNase) not only reduces the immune effect but also increases plasma half-life. Nonetheless, the available PEG-ASNase is randomly pegylated and, consequently, with high degree of polydispersity. In this work we developed a site-specific N-terminus pegylation protocol for ASNase using methoxy-polyethylene glycol carboxymethyl N-hydroxysuccinimidyl ester (mPEG-NHS, MW = 10 kDa). Reaction parameters investigated were ionic strength, pH and reaction time to optimize yield and minimize polydispersity. Results confirmed pegylation and low polydispersity. Buffer ionic strength favored N-terminal conjugation at 100 mM phosphate buffer saline (PBS). Additionally, pH and reaction time were found to increase polyPEGylated species. The highest yield of monoPEG-ASNase, 42%, corresponded to reaction conditions of pH 7.5, reaction time of 30 min and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was purified in a strong anionic column with NaCl linear gradient up to 170 mM and monoPEG-ASNase was eluted with 78 mM NaCl, 70% pure (55% yield). Size exclusion chromatography was further performed and increased monoPEG-ASNase purity to 99% (45% final yield). The monoPEG-ASNase, 190 ± 10 kDa was stable for 21 days at 4ºC while nonpegylated ASNase (control), 146 ± 6 kDa, lost 52% of its activity within 7 days at 4ºC. Enzyme kinetics were studied and similar kM values were obtained for both native and monoPEG-ASNase, 20 µM and 35 µM respectively. The monoPEG-ASNase demonstrated to be resistant to the plasma proteases asparaginyl endopeptidase and cathepsin B, 84 h at 37 °C and also was cytotoxic to leukemic cells (MOLT-4 and Reh). Therefore, site-specific N-terminus pegylation of ASNase resulted in a novel enzyme variant with preserved catalytic activity and, therefore, promising for the treatment of LLA.

Protein PEGylation is the covalent bonding of polyethylene glycol (PEG) polymers to amino acid residues of the protein and it is one of the most promising techniques for improving the therapeutic effect of biopharmaceuticals and long-term stability of protein-based biosensors. This chemical modification brings advantages to biopharmaceuticals, such as an increased half-life, enhanced stability, and reduced immunogenicity. Moreover, in the analytical field, PEGylation improves the multiple properties of protein-based biosensors including biocompatibility, thermal and long-term stability, and solubility in organic solvents. However, the use of PEGylated conjugates in the analytical and therapeutic fields has not been widely explored. The limited industrial application of PEGylated bioconjugates can be attributed to the fact that the reaction and separation steps are currently a challenge. The correct selection of the PEGylation reaction design and the purification process are important challenges in the field of bioconjugation. In this sense, the design and optimization of site-specific PEGylation reactions and application of aqueous biphasic systems (ABS) as purification platforms for PEGylated conjugates are the two main objectives of this thesis. Regarding the purification step, the efficient fractionation (i) of the PEGylated conjugates from the native protein and (ii) of the PEGylated conjugates based on their degree of PEGylation was studied. Centrifugal partition chromatography (CPC) was applied as a continuous regime platform based on ABS technology to efficiently purify the PEGylated proteins. The two proteins under study are L-asparaginase, an important biopharmaceutical applied in the treatment of acute lymphoblastic leukemia and cytochrome c, a promising biosensor. The current work developed in this thesis demonstrates the great potential of ABS in the fractionation of PEGylated proteins, under batch and continuous regime. In addition, in situ recovery of the PEGylated products through one-pot bioconjugation and ABS purification was successfully demonstrated for both enzymes studied. Although further research on scale-up is still required, the results presented show the relevance of ABS platforms for the development of separation processes of PEGylated proteins. / A PEGuilação de proteínas é a ligação covalente de polímeros de polietilenoglicol (PEG) a resíduos de aminoácidos da proteína e é uma das técnicas mais promissoras para melhorar o efeito terapêutico dos biofármacos e a estabilidade a longo prazo de biossensores proteícos. Esta modificação química traz vantagens aos produtos biofarmacêuticos, como um aumento da meia-vida, maior estabilidade e imunogenicidade reduzida. Além disso, no campo analítico, a PEGuilação melhora as múltiplas propriedades dos biossensores baseados em proteínas, incluindo biocompatibilidade, estabilidade térmica e a longo prazo, e solubilidade em solventes orgânicos. No entanto, o uso de conjugados PEGuilados em campos analíticos e terapêuticos não tem sido amplamente explorado. A aplicação industrial limitada dos bioconjugados PEGuilados pode ser atribuída ao facto de as etapas de reacção e separação serem atualmente um desafio. A seleção correcta do design da reacção de PEGuilação e do processo de purificação são importantes desafios no campo da bioconjugação. Neste sentido, a concepção e otimização de reações de PEGuilação sítio-específicas e aplicação de sistemas aquosos bifásicos (ABS) como plataformas de purificação de conjugados PEGuilados são os dois principais objetivos desta tese. No que concerne à etapa de purificação foi estudado o eficiente fracionamento (i) dos conjugados PEGuilados, da proteína nativa e (ii) dos conjugados PEGuilados baseados no seu grau de PEGuilação. A cromatografia por partição centrífuga (CPC) foi aplicada como uma plataforma de regime contínuo baseada na tecnologia de ABS para purificar eficientemente as proteínas PEGuiladas. As duas proteínas em estudo são a L-asparaginase, importante biofármaco aplicado no tratamento da leucemia linfoblástica aguda e o citocromo c, um potencial biossensor. A partir dos trabalhos desenvolvidos, é possível confirmar o grande potencial dos ABS no fracionamento de proteínas PEGuiladas, em regime contínuo e descontínuo. Além disso, a recuperação in situ dos produtos PEGuilados através da integração em uma única etapa de bioconjugação e purificação por ABS foi comprovada com sucesso para ambas as enzimas estudadas. Embora ainda sejam necessários estudos adicionais sobre a viabilidade destes sistemas em larga escala, os resultados aqui apresentados demonstram a relevância dos ABS para o desenvolvimento de processos de separação de proteínas PEGuiladas.

Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.

In the present thesis we propose the development of hybrid polymer titanium dioxide (TiO2) nanoparticles for use in biomedical applications. TiO2 exhibits high biocompatibility in the dark however, upon illumination in aqueous media with near UV light it produces an array of reactive oxygen species (ROS) which have the capability to induce death in neighbouring cells. The process of inducing cell death using a photosensitive material which produces ROS is known as photodynamic therapy (PDT) and is used to treat a wide range of maladies from psoriasis to cancer.We have demonstrated the ability to produce anatase nanoparticles with high control over their resulting size through a novel water mediated sol-gel synthetic method in benzyl alcohol, using either Ti(OnPr)4, Ti(OnBu)4 or Ti(OiPr)4 as the metal precursor. Through dynamic light scattering (DLS) analysis we have shown that the mechanism of nanoparticle growth appears to proceed through the agglomeration of primary nanoparticles formed instantly upon adding the reagents together. After synthesis the nanoparticles could be easily redispersed in aqueous media at pH2 with any further agglomeration being controlled by the parent alkoxide.After synthesis the nanoparticles were coated with PEG, conjugated to either a catechol or phosphate as ligand, in order to stabilise the nanoparticles at neutral pH. Uncoated nanoparticles exhibited good photoactive capability in the photooxidation of methylene blue. However, on coating with catechols the photoactivity of the nanoparticles was abolished. Coating with phosph(on)ates on the other hand preserved or even enhanced the photoactivity which makes this system promising for in vivo applications.At the same time this thesis also reports preliminary investigations on the use of TiO2 embedded into the walls of model drug loaded poly electrolyte multilayer microspheres for UV triggered delivery applications.