Rozprawy doktorskie na temat „Biomedical and chemical applications”

Aromatic and aliphatic diacid chlorides were used to condense naturally occurring diamino acids and their esterified derivatives. It was anticipated the resulting functional polyamides would biodegrade to physiologically acceptable compounds and show pH dependant solubility could be used for biomedical applications ranging from enteric coatings to hydrosoluble drug delivery vehicles capable of targeting areas of low physiological pH. With these applications in mind the polymers were characterised by infra red spectroscopy, gel permeation chromatography and in the case of aqueous soluble polymers by potentiometric titration. Thin films of poly (lysine ethyl ester isophthalamide) plasticised with poly (caprolactone) were cast from DMSO/chloroform solutions and their mechanical properties measured on a Hounsfield Hti tensiometer. Interfacial synthesis was investigated as a synthetic route for the production of linear functional polyamides. High molecular weight polymer was obtained only when esterified diamino acids were condensed with aromatic diacid chlorides. The method was unsuitable for the production of copolymers of free and esterified amino acids with a diacid chloride. A novel miscible mixed solvent single phase reaction was investigated for production of copolymers of esterified and non-esterified amino acids with diacid chlorides. Aliphatic diacid chlorides were unsuitable for condensing diamino acids using this technique because of high rates of hydrolysis. The technique gave high molecular weight homopolymers from esterified diamino acids and aromatic diacid chlorides.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 153-164).
Functional polymeric thin films are often stratified with nanometer level structure and distinct purposes for each layer. These nanostructured polymeric materials are useful in a wide variety of applications including drug delivery, tissue engineering, controlling condensation and polymeric batteries; all of which will be discussed in this work. The first area of my thesis will detail the use of C₆₀ cluster-ion depth profiling X-ray Photoelectron Spectroscopy (XPS) to fundamentally understand how thin film structure and function relate. This method has the unique capability to determine the atomic composition and chemical state of polymeric thin films with <10nm nanometer depth resolution without any chemical labeling or modification. Using this technique, I probed the nanostructure of functional thin films to quantify the interlayer diffusion of the biopolymer chitosan as well as demonstrate methods to stop this diffusion. I also explored the role of interlayer diffusion in the design of hydrophobic yet antifogging 'zwitter-wettable' surfaces. Additionally, I probed the lithium triflate salt distribution in solid block copolymer battery electrolytes (PS-b-POEM) to understand the lithium-ion distribution within the POEM block. In the second area of my thesis, I show how the nanostructure of materials control the function of polymeric particles in vitro and in vivo. One example is a 'Cellular Backpack' which is a flat, anisotropic, stratified polymeric particle that is hundreds of nanometers thick and microns wide. In partnership with the Mitragotri group at UCSB, we show that cellular backpacks are phagocytosis resistant, and when attached to a cell, the cell maintains native functions. These capabilities uniquely position backpacks for cell-mediated therapeutic delivery and we show in vivo that immune cells attached to backpacks maintain their ability to home to sites of inflammation. In addition, we have designed polymeric microtubes that can control their orientation on the surface of living cells. Inspired by chemically non-uniform Janus particles, we designed tube-shaped, chemically non-uniform microparticles with cell-adhesive ligands on the ends of the tubes and a cell-resistant surface on the sides. Our results show that by altering the surface chemistry on the end versus the side, we can control the orientation of tubes on living cells. This advance opens the capability to control phagocytosis and design cellular materials from the bottom up.
by Jonathan Brian Gilbert.
Ph. D.

Switchable oligopeptides, able to expose of conceal biomolecules on a surface, upon the application of an electrical potential, represent a versatile tool for the development of novel devices, presenting potential biomedical applications. Recently, several studies have demonstrated the applicability of smart devices for the control of protein binding and cellular response. In this work; a detailed analysis of the steric requirements necessary to develop a mixed oligopeptide Self-Assembled Monolayer (SAM) presenting an optimum switching ability will be described. The influence of both the SAM components surface ratio and the switching unit length on the mixed SAMs switching performance will be investigated. The findings of this investigation will be used to develop, for the first time, a platform, based on electrically switchable oligopeptides, able to control the interaction between an antigen and its relative antibody. The influence of the biological medium on the oligopeptide switching ability will also be investigated. Finally, an orthogonal functionalisation strategy, will be investigated in detail, together with a new platform able to promote human sperm cells adhesion. The results of this research thesis will also represent the first building blocks towards the development of glass-gold rnicropattemed surfaces able to control the calcium signalling in human sperm cells, presenting potential applications in the improvement of in-vitro fertilisation (NF) treatments success rates.

Ion sensitive field effect transistors (ISFETs) have long been used as analogue chemical sensors particularly for biomedical applications. However, there are some applications where a "yes" / "no" type answer regarding pH change is sufficient. For example, in DNA sequencing the question is whether a chain extension reaction took place or not. Detecting this at the sensing point reduces the sensing process to pH change threshold detection. It eliminates the need for analogue to digital conversion and facilitates an all digital sensory system. This thesis presents Novel Floating Gate ISFET based Chemical Inverters that were created with semiconductor based biomedical applications in mind. It starts by allowing two ISFETs to share the same ion sensing membrane and a common floating gate. Arranging them in a simple FG inverter configuration, their switching may be triggered by either the reference voltage or chemical pH change. In order to enhance its input noise immunity, a chemical Schmitt Trigger is presented. Using ISFETs for the detection of minute pH changes have been a challenge. A simple method to locally scale input referred chemical signal at the ISFET's floating gate is presented. It is based on using the ratio of capacitive coupling to the floating gate. The chemical signal is coupled via the passivation capacitance (Cpass) while an electrical input (V2) is coupled via a poly capacitance (C2). V2 sees the chemical signal with a scaling of Cpass/C2, which can be designed. Finally, ISFETs suffer from initial trapped charges that cause mismatch between devices in the same die. A fast matching method is presented here, that can be used to hugely reduce mismatch of arrays of FG devices. It is based on using indirect bidirectional tunnelling. Two tunnelling structures are added to each ISFET's FG, one adds electrons to it while the other removes them. It is possible to match all ISFETs' initial FG voltages to a point where both tunnelling currents reach equilibrium.

This thesis presents the design and characterization of a novel pulsed miniature capacitively-coupled Atmospheric Pressure Glow Discharge Torch (APGD- t) aimed at localized biomedical applications. Amplitude modulation of the 13.56 MHz carrier signal allows to continuously vary the power level applied to the APGD-t. Typically, the APGD-t produces a plasma jet with a 150-500 μm diameter and ≈2.5 mm length. Helium (He) is the plasma-forming gas with a flow rate ranging from 0.5 to 1.5 slm. The use of a small capillary electrode enhances the electric field, lowering the breakdown voltage (typically 220 Vpk-to-0) and allows the injection of small amounts (0-50 sccm) of a source of reactive species (O2) downstream of the plasma-forming region, in the plasma afterglow. The O2 is electronically dissociated in the plasma afterglow to create atomic oxygen (O) with no effect on the electrical properties. A ratio of 0.3% v/v, O2/He generates a maximum in O production.
Careful electrical probe measurements and circuit analyses reveal the strong effect of commercial passive voltage probes on the total load impedance of the APGD-t circuit. The larger the probe capacitance and cable length, the larger the component of the phase angle between the load voltage and circuit current signals induced by the probe. The calibration of the phase angles induced by the voltage probes allows to estimate that a resistive power of ~0.24-1 W is dissipated in the APGD- t under nominal operating conditions.
The gas kinetic and atomic He excitation temperatures, and the electron density near the APGD-t nozzle exit are estimated at ≈323 K, ≈1914 K and ≈1011 cm-3, respectively. This confirms that the APGD-t plasma jet near the nozzle exit is in a non-thermal equilibrium state. The emission spectroscopy study reveals the entrainment of air molecules (N2, O2 and H2O) in the plasma jet, and that their excitation by the plasma creates new reactive species (O and OH). A preliminary survey of the chemical reactions taking place in the plasma afterglow reveals that metastable He as well as OH, O, O2(a1Δg), O2(b1Σg+), N2, N2+ and O3 are plasma species that can reach and react with organic or biological surfaces located a few mm downstream of the APGD-t nozzle exit. This thesis demonstrates that the APGD-t is a promising tool for localized biomedical applications.

Hydrocolloid materials have been used for some time in the fields of regenerative medicine and drug delivery. Despite a significant body of work, to date the majority of research in the area has focused on relatively simple compositions and microstructures. In comparison, the food industry has long used refined and often subtle methods to structure and thereby tailor the release and handling properties of a vast range of similar materials. In this thesis, a range of processing methodologies has been used to generate novel materials intended for use in the regenerative medicine and drug delivery using gellan and kappa carrageenan. The thesis demonstrates how even small changes in process conditions can result in significant changes in the way a material handles and may deliver therapeutic molecules. This thesis has demonstrated that gellan can be used to form robust quiescent structures, as well as shear thinning fluid materials by changing the processing and formulation. Furthermore, it was demonstrated that it was possible to generate a novel cell delivery device by the hydration of kappa carrageenan in warm biomedical buffers. Overall this thesis demonstrates the range and complexity of structures that can be produced using the relatively small number of polymers.

The main objective of this dissertation is the synthesis and study of modified surface systems for the development of bioactive platforms and their use in specific biotechnological and biomedical applications. This work has led to various biological template development projects; all in attempts to provide new surfaces and probes in nanotechnology. These projects focus mainly on protein modified surface platforms, liposome based spherical platforms, and carbon nanotubes based magnetic platforms. The planar platforms include gold, silicon and aluminum oxide surfaces. Spherical surfaces such as liposomes and nanoparticles were also studied, and finally, surface modification was extended to carbon nanotubes and magnetic nanoparticles. In this dissertation, the planar surface work focuses on demonstrating the behavior of proteins at interfaces in terms of conformation, stability and activity (e.g., of avidin, trypsin and antibodies) using fluorescence microscopy. Different ligands were attached chemically on the surfaces to incorporate hydrophobic hydrophilic and charged characteristics. A chelating agent (iminodiacetic acid, IDA), an affinity ligand (biotin), and reactive groups (amino and carboxylic groups) were covalently incorporated onto the surfaces. Proteins including myoglobin, cytochrome C, avidin, trypsin and immunoglobulin G (IgG) were used in this study. The results show that proteins and ligands were successfully attached to different surfaces. Protein adsorption studies illustrate activity decrease by using fluorescence intensity. After attachment on hydrophobic functionalized surfaces. Along the same line, experiments were conducted on the comparison of silicon dioxide and gold-coated surfaces with immobilized enzymes, small molecules, and polymers for potential use as biosensors. Silicon dioxide wafers were prepared via silanization with 3-aminopropyl triethoxysilane (APTES) followed by glutaraldehyde activation and, finally, protein and/or small ligand attachment. Gold-coated surfaces were utilized for immobilizations using 16-mercaptohexadecanoic acid (MHA) which forms self-assembled monolayers (SAMs) on gold surfaces followed by covalently attachment of proteins. The activity of trypsin immobilized onto these surfaces was also measured. The silicon dioxide wafers when modified first with NH₂-PEG-NH₂ allowed for trypsin a relatively higher activity with about 11% greater activity than when attached on gold surfaces and 84% higher activity than on bare silicon surfaces. Furthermore, the bimolecular silicon dioxide surfaces were shown to be much more stable than the gold surfaces. The silicon dioxide surfaces with an immobilized reversible inhibitor, p-aminobenzamidine (PAB), show to very effectively bind proteins from solution compared to gold surfaces. Liposome were studied because their versatility and vast implications in bio-sensing and drug-delivery potential. In this work, liposomes were prepared with the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. The amino groups of DMPE were then modified with ligands that included iminodiacetic acid (IDA), and PEG. These functionalized liposomes were used to prepare dispersed gold “nano-dots” on their surface. These novel functional liposomes, with chelating ligands and polymers can be used to bind biomolecules and active compounds (nanoparticles of gold, quantum dots, drugs) with long stability. The results show that we can successfully manufacture functional liposomes and form gold nanoshells on their external surface. These two types of systems can be used as drug delivery, and as imaging systems. Their characterization and potential use in biomedical applications as contrast agents seems quite promising once complexity and stability of these gold nanoshells is elucidated. The modification and preparation of functional-carbon nanotubes was investigated with the chemical hetero-junction analysis between magnetic nanoparticles coated poly-acrylic acid (PAA) and multi-wall carbon nanotubes (MWCNTs). Magnetic nanoparticles were covalently attached to open-ended nanotubes. Initial evidence suggests that short functionalized multi-wall nanotubes can be continuously connected at their terminal ends for build-up of relatively large nanostructures based on serial configurations. It is shown that magnetic carbon nanotubes systems exhibit defined arrangements due to the influence of magnetic fields. Indeed, linear arrays of carbon nanotubes inter-connected through magnetic nanoparticles were prone to be manipulated in the presence of a magnet device. A potential application of these magnetic nanostructures was shown by successfully manipulating agarose beads in buffer solution as a model system. These results suggest that the use of continuously connected magnetic nanostructures with non-modified sidewall surfaces will find potential applications in the areas of bio-sensing, force transduction and cancer screening-manipulation among others.

Mestrado em Ciência e Engenharia de Materiais
Calcium phosphate-based materials, in particular hydroxyapatite-based ones,are among the most important materials for biomedical applications (bone graftsubstitutes, drug delivery systems, etc.). Owing to their compositional similaritywith respect to hard tissues, these materials show superior bioactive,osteoconductive, cell seeding and growth environment properties. Additionally,their capability to adsorb biological important substances like proteins, drugs,etc. makes them interesting materials to be used as drug delivery systems. Several studies on the effects of morphological aspects like particle size,shape, pore size and pore volume on the biological behaviour of calciumphosphate-based materials have shown that the properties of these materialscannot be considered merely on compositional aspects, but the role ofmorphological issues must also be taken into consideration. In the present work, calcium phosphate particles with a wide range of sizeswere produced by precipitation in calcium/citrate/phosphate solutions. It wasobserved that the manipulation of experimental conditions, namely the citrate-calcium ratio (Cit/Ca) and the pH of the solution, allowed to producehydroxyapatite particles either as nanosized particles, either as micrometricsized aggregates with particular shapes. The different sizes and shapes wereanalyzed in the framework of nucleation and growth phenomena and henceattributed to the development of different particle surface charge conditionsrelated to the adsorption of differently charged citrate species. The study of the preparation of calcium phosphate porous granules by spraydrying the suspensions of the various precipitated hydroxyapatite particles wasalso undertaken in the present work. The obtained results showed that thedifferent morphologies of the suspended hydroxyapatite particles havesignificant effects on the spray dried granules’ morphology and microstructure,thus accounting for different pore size and pore size distributions. Moreover,the study of the spray dried granules heat treatment demonstrated that not onlythe granules’porosity may be further modified but also its crystal phasecomposition. In view of the potential applications of the porous materialsprepared in this work such as drug, growth factors and stem cells carriers or aspromoter of cell adhesion, the present study points out to a wide range ofpossibilities for producing calcium phosphate porous granules with a differentschedule of morphological characteristics.
Os materiais fosfo-cálcicos, particularmente aqueles à base de hidroxiapatite, são dos mais importantes para aplicações biomédicas, como por exemplo, a substituição óssea e os sistemas de libertação controlada de fármacos. Este facto deve-se principalmente à semelhança da sua composição com a parte inorgânica do tecido ósseo. É esta semelhança que está na origem dasnotáveis propriedades biológicas destes materiais, tais como: excelente bioactividade e osteoconductividade. Por outro lado, estes materiais possuem ainda a capacidade de adsorver substâncias com interesse biológico,(proteínas, drogas, etc.) o que os torna interessantes como sistemas delibertação controlada de fármacos. No entanto, alguns estudos têmdemonstrado que o comportamento biológico dos materiais fosfo-cálcicos não depende apenas da sua composição mas também de aspectos morfológicos, tais como: tamanho e forma departícula, tamanho e volume de poro, etc. No presente trabalho produziram-se, por precipitação a partir de soluções de cálcio/citrato/fosfato, partículas de fosfato de cálcio com uma grandediversidade de tamanhos. Observou-se que a manipulação das condiçõesexperimentais, nomeadamente a razão citrato/cálcio (Cit/Ca) e o pH dasolução, possibilitaram a produção de partículas de hidroxiapatite, quer na forma de partículas com tamanhos nanométricos, quer na forma de agregados micrométricos com formas peculiares. A variedade de tamanhos e formas daspartículas produzidas foi analisado no contexto dos fenómenos de nucleação e crescimento, tendo sido atribuídaao desenvolvimento de diferentes condições de carga superficial devidas à adsorção de espécies iónicas de citrato com diferentes cargas. No presente trabalho desenvolveu-se também o estudo da preparação de grânulos porosos de fosfato de cálcio, por atomização de suspensões de partículas de hidroxiapatite com diferentes morfologias. Os resultados obtidosmostraram que a utilização de partículas com diferentes morfologias influenciasignificativamente a morfologia e microestrutura dos grânulos atomizados, oque origina grânulos com diferentes tamanhos e distribuição de tamanho deporos. Além disso, demonstrou-se que o tratamento térmico permite modificar não só a porosidade dos grânulos, mas também a sua composição cristalina.Tendo em vista as potenciais aplicações dos materiais porosos preparadosneste trabalho, tais como sistemas de libertação controlada de fármacos,factores de crescimento e de células estaminais ou como promotores daadesão de células, o presente trabalho sugere a possibilidade de produção de grânulos de fosfato de cálcio com uma vasta multiplicidade de características morfológicas.

Engineered biological systems are increasingly used to produce fuels, pharmaceuticals and industrial chemicals. While transforming cells into renewable chemical factories presents an enormous opportunity, development timelines are long, costly and often uncertain. Engineering microbes for chemical production is accomplished through the biological design-build-test cycle: many designs are formulated, the corresponding organisms are constructed, and their ability to produce the desired chemical is evaluated. Designs that perform well become the starting point for the next round of the cycle. Faster design cycles result in shorter and less costly product development timelines. Advances in DNA sequencing, synthesis and genome engineering technologies have sped up the design and build steps of the design cycle by enabling billions of organism variants to be designed and constructed simultaneously. However, evaluation of the resulting designs continues to rely on low-throughput technologies with evaluation rates on the order of thousands per day. Because the engineering process is a cycle, it can only proceed at the rate of the slowest step. A high-throughput method for design evaluation would increase the throughput of the design cycle by up to a million-fold. This thesis describes an engineering framework that makes high-throughput design evaluation a reality. By programming cells to keep track of their own success in making a desired product, I enable screens and selections to be used for the optimization of metabolic pathways. I develop biosensors that maintain gene expression at a rate proportional to the concentration of several different chemical products and show that higher product concentration results in a higher fluorescent output. I then construct metabolic pathways for the production of the renewable plastic precursors 3-hydroxypropionate, acrylate, glucarate and muconate. I combine each pathway with the appropriate biosensor and use fluorescence to observe product formation in real-time. Next, I replace the fluorescent protein with an antibiotic resistance gene and link the level of product formation to the cell’s ability to survive an antibiotic challenge. I deploy the selection to optimize production of both glucarate and naringenin from glucose. I further develop the characterization of these new biosensors to promote their use as genetic switches for synthetic biological circuits. Finally, I develop a device called the fluorimostat that makes long-term closed-loop programmable control of gene expression a reality.
Engineering and Applied Sciences - Engineering Sciences

Present PhD thesis aimed to investigate relatively unknown properties of halloysite nanoparticles, as well as to further examine HNTs as potential drug nanocarriers. NPs loading and release characteristics were studied using model active molecules: magnesium monoperoxyphthalate (MMPP), aspirin and epirubicin. The research was fulfilled with formation of complex multi-functional nanoarchitectures, which apart from ability to deliver incorporated drugs, showed the potential of controlled and sustain release of therapeutics, biocompatible and bioresorbable characteristics as well as potential targeting abilities. Great attention was dedicated to characterization of formed halloysite-based nanoarchitectures in qualitative as well as quantitative manner. Investigations performed in this thesis also faced the problem of exceeding dimensions of halloysite units, nanoparticles aggregation, poor loading capability and dose dumping effect. Subsequently, studies for trying to find a solution to these obstacles were undertaken. Fully characterized halloysite nanoconstructs were further examined in biological field, employing different cancer cell lines. Studies on pristine halloysite nanotubes: Physico-chemical and biological properties of halloysite nanoparticles were evaluated using microscopic techniques, spectroscopic analysis, surface studies regarding charge, porosity and wettability. The thermal and time-based examination of pristine halloysite was performed as well, showing stability of HNTs alumino-silicate skeletons up to ~400 ℃ and over a long period of time (2 years) at room temperature, however with a variable amount of incorporated water molecules. Biological performance of HNTs was determined in vitro in multiple cellular systems by toxicity, cellular uptake, colocalization and accumulation studies using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction (MTT) assay and set of microscopic techniques. Aiming to deeply characterize halloysite nanoparticles, the study proceeded with employment of non-standard techniques, as multiphoton microscopy that drove to discovery of novel NPs promising capabilities. It was revealed that halloysite is able to convert light to its second harmonic, at twice of the frequency (and therefore half of the wavelength) while using high intensity femtosecond pulsed laser. Halloysite Second Harmonic Generation (SHG) signal was detected over a broad wavelength range, showed stability over a long period of time, polarization properties and quadratic dependence on the intensity of incident light. The analysis also pointed out characteristic structure properties of the nanoparticle that is lack of the center of symmetry and the high crystalline structure organization. Among a wide spectrum of domains where discovered HNTs characteristics can be utilized (e.g. optoelectronics, biosensors), we have explored its application in alternative label-free bioimaging. The proposed multiphoton method of analysis showed advantages over the standard confocal microscopy, since e.g. nanoparticles did not have to be stained prior the analysis, thus no possible alterations of HNTs including size, surface chemistry and consequent cellular uptake were induced. Therefore, for the first time, halloysite nanotubes were exploited as imaging agents, taking advantage of their endogenous properties. Along the research it was revealed that the length of pristine HNTs and the strong aggregation limit their ability to pass intracellular membranes and thus minimize their effectiveness as drug nanocarriers. Therefore, efforts were devoted to the development of facile methodology to efficiently disperse and shorten HNTs units. Set of characterizations techniques, such as Scanning Electron Microscopy (SEM) analysis with size distribution profile and nitrogen adsorption Brunauer–Emmett–Teller (BET) method revealed that the applied ultrasonication procedure resulted with longest tubes breaking and favored obtaining HNTs below 300 nm in length (39.1 % to 76 % of the batch). The number of voids among the pristine nanoparticles when packing together (123–43 nm) greatly increased (total pore volume from 0.23 cm^3/g to 0.30 cm^3/g), meaning that the nanomaterial was efficiently disaggregated as well. In vitro internalization and colocalization studies by Scanning Electron and Multiphoton Microscopy demonstrated that the sonicated halloysite were preferentially internalized via macropinocytosis within 60 min and accumulated in the perinuclear region within 24 h. Halloysite application in nanomedicine: To study halloysite potential as a carrier for drugs, we set up the preparation and characterization of hybrid nanoconstructs with model molecules such as magnesium monoperoxyphthalate hexahydrate (MMPP), a negatively charged oxidizing agent, aspirin, an anti-inflammatory drug and epirubicin, a chemotherapeutic. Chosen molecules were incorporated with 3.5 %wt, 1.1 %wt and 5.1 %wt capacity, respectively for MMPP, aspirin and epirubicin. Loading efficiency (LE) improvement was achieved through the choice of the right solvent (1), enhancement of electrostatic forces between nanoparticle and the drug, via functionalization of HNT surfaces with an active linker (2), as well as NPs structure modification leading to increase of inner lumen volume (3). Specifically, the use of water: EtOH (7:3 v/v) as a solvent instead of water, increased MMPP loading capacity up to 6.1 %wt. Poor incorporation of aspirin was improved by enhancing electrostatic forces between deprotonated aspirin molecules and modified HNTs inner walls with amine-rich organosilane. It was also demonstrated that by enlarging volume of the NPs cavities, more molecules could be loaded. To do that, pristine HNTs were treated with 0.1M aqueous solution of NaOH, which resulted in an exfoliation of bilayers located inside the lumen. At the same time, outer surface of the halloysite tubules was preserved. As a consequence of the base treatment, halloysite cylinders gained more volume in the inner cavity as concluded from Transmission Electron Microscopy (TEM) and nitrogen adsorption BET analysis. The actual test on loading capacity using model MMPP molecule revealed increased MMPP incorporation from 6.1 % wt to 11.7 %wt. To evaluate if the activity of MMPP as an oxidizing agent remained unchanged upon incorporation and release from halloysite, and therefore to demonstrate the inactivity of the inorganic skeleton towards carried molecule, we tested HNT-MMPP nanoconstruct with selective fluorescent 1,3-diphenylisobenzofuran (DPBF) probe. Among available modifications of halloysite nanoparticles via covalent bond, the surface silanization is commonly recognized as one of the most efficient and widespread reaction while HNT manipulation. Up to date, the halloysite nanotubes functionalized with silanes have been used as a support for versatile applications in diverse scientific domains, including enzymes immobilization and biosensing. Willing to explore the halloysite functionalization with those active linkers, we have performed grafting reactions with representative organosilanes carrying the same backbone, while varying in the content of terminal groups, namely (3-aminopropyl)triethoxysilane (APTES), 3-(2-aminoethylamino)propyldimethoxymethylsilane (AEAPS), (3-mercaptopropyl)trimethoxysilane (MPS). Successful HNTs surfaces functionalization with organosilanes was demonstrated by means of quantitative thermogravimetric analysis (TGA) that allowed to estimate the loading capacity of organosilanes to be of 5.7 %wt for APTES, 7.4 %wt for AEAPS and 0.7 %wt for MPS. In addition, particular attention was dedicated to further quantify incorporated organosilane (APTES), since only one method has been so far reported, that is the destructive thermogravimetric analysis (TGA). For this reason, we set up and optimized a Fmoc based method by performing the following three reactions: (i) synthesis of “APTES-Fmoc” molecule; (ii) halloysite functionalization with “APTES-Fmoc”; and (iii) time-dependent Fmoc deprotection reaction in piperidine: EtOH (20 %) solution, resulting in dibenzofulvenepiperidine adduct (DBF-pip) formation. The UV-visible spectroscopic analysis of supernatant solutions demonstrated that the DBF-pip deprotection from halloysite support needs 5 h to be completed. Therefore, it was evidenced that HNT Fmoc-method showed strong coherence with already existing TGA method (± 2 % measurement error) and stood out as a valuable complementary technique for quantification of silane grafting on HNTs surface with additional low-cost and nondestructive advantages. The possibility of using halloysite nanotubes as a non-viral gene delivery nanosystem for therapeutic treatments was studied as well. Aiming to immobilize plasmid DNA (pDNA) based on the Green Fluorescent Protein (GFP) on HNTs support, the layer-by-layer (LbL) adsorption technique was applied. Obtained multi-component assembly was characterized qualitatively by monitoring variation in nanoparticle physico-chemical properties including surface charge, mass weight, presence of functional groups at each step of hybrid formation, which confirmed the successful nanoarchitecture formation. In order to additionally demonstrate the presence of GFP encoding plasmid (pGFP) on HNTs, the nanoarchitecture was treated with the bovine pancreatic deoxyribonuclease (DNase) enzyme, which induced the pGFP degradation through hydrolytic cleavage of phosphodiester linkages in DNA backbone. Thus, as expected, such nanoform with deposed genetic material varied in physico-chemical properties, expressing similar ones of the nanoconstruct without pGFP plasmid attached. The biological efficiency of HNTs-pGFP nanosystem was checked by means of Multiphoton microscopy. Successful pGFP plasmid transportation into cells was verified by detection of GFP expression, which yielded fluorescence emission. The interesting and innovative aspect of this case study was the simultaneous observation of GFP expression via fluorescence detection, and colocalization of halloysite nanoparticles by their SHG signal. This study proved that halloysite can act as an efficient carrier of genetic material, since free pGFP cannot be internalized by same cells, due to its large size and significant charge. Drug-loaded halloysite nanoconstructs (HNT-MMPP, HNT-APTES-aspirin) were also examined on the drug release kinetics, demonstrating long-term MMPP leakage taking 18 days and aspirin over 60 min. However, great drug liberation into the solvent of release was observed in the first minutes, followed by desired sustained drug release. The initial molecule liberation (dose dumping effect) is known to entail local toxicity. Herein, trying to find a solution to this problem, the coating of HNTs with the natural collagen polymer was investigated. Two strategies for the loading with this biopolymer were studied: (i) formation of a covalent bond between collagen and APTES-modified HNTs using glutaraldehyde cross-linker or (ii) noncovalent adsorption of collagen into pores of NPs. Immobilization of collagen on the surface of HNTs was estimated to be 3.7 %wt (i) and 1.8 %wt (ii). Other supplementary characterization techniques, such as water contact angle, ζ–potential analysis, Kaiser test, ultraviolet and visible (UV-vis) spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR) were in accordance and proved nanoarchitectures formation. For the visualization purpose of HNTs encapsulated in collagen shell, the innovative characterization technique was implemented, namely 3D Multiphoton microscopy. It revealed that the biopolymer coating blocked the entrances of the hollow tubes thus, entrapping the drug in NPs. Mimicking tumor microenvironment (TME), the pH and/or enzyme triggered release was performed. LC-Mass analysis revealed that the collagen coating slowed down the release of aspirin from HNTs. Studies on cells showed that the collagen coating on HNTs is biocompatible and cell viability assay performed on 5637 urinary bladder and HeLa cervical cancer cell lines demonstrated the sustained release of the entrapped epirubicin chemotherapeutic agent in the biological context. Industrial application of halloysite: During a stage in BASF SE (USA), validation and properties enhancement of halloysite-based products potentially manufacturable in the company on an industrial scale were studied. In particular, the research was dedicated to aspects such as the pH-dependent dispersion behavior of halloysite nanotubes and iron coarse impurities removal from bulk samples. Applied methodologies and set of physico-chemical characterization techniques generated and revealed decreased percentage of present aggregates, maintained low shear viscosity under the threshold value and increased solids loading capacity in final halloysite-based products. Conclusions: In conclusion, PhD studies here reported contributed to the exploration of halloysite nanotubes for their application in the nanomedical and industrial fields. The investigations suggest a facile manipulation and functionalization of HNTs, useful for properties modification and improved NPs performance. Specifically, the study was directed toward formation of multi-functional nanocarriers with controlled drug delivery and release properties, together with targeting and imaging abilities. Moreover, the research was completed with halloysite-related technology transfer to the BASF SE, for the purpose of knowledge increase in the halloysite-field and bringing forward placement of halloysite-based products on the market. The systematic study on HNTs characterization and application performed in this PhD thesis will contribute to the development of HNTs as a high performance structural and functional material.

Imaging of biomolecules in biological substrates by mass spectrometry or spectroscopic imaging techniques plays a major role in biomedical, clinical, and pharmaceutical research. The work presented in this thesis investigates the capabilities of three imaging techniques, liquid extraction surface analysis (LESA) mass spectrometry imaging (MSI), matrix assisted laser desorption ionisation (MALDI) MSI and stimulated Raman scattering (SRS) microscopy. A method for combined LESA and MALDI analysis was developed and results provided high resolution imaging of multiple analyte classes (proteins, lipids and small molecule drugs) in thin tissue sections. SRS microscopy was used for the quantitative imaging of MALDI sampling effects and sample preparation, providing insight into fundamental processes of MALDI MS. Multimodal SRS, LESA and MALDI imaging was executed on a single tissue sample revealing the complementarity between the three approaches. Specific challenges for LESA were further explored, namely quantification, improved spatial resolution and alternative biological substrates. A quantitative LESA method based on the production of mimetic tissue models containing stable isotope-labelled proteins was developed. An alternative platform, the Flow-Probe™, with the potential to achieve higher spatial resolution was assessed. Finally, a LESA method for the direct analysis of proteins from live bacterial colonies was developed.

The potential of magnetic nanoparticle-vesicle assemblies (MNP-V) as remote controlled drug delivery platforms capable of inducing cellular responses under magnetic stimuli has been previously demonstrated in the Webb group at the University of Manchester. To create these magnetoresponsive nanomaterials biotin-avidin and Cu-histidinyl multivalent recognition were employed. This thesis describes an exploration of the potential of thiol-thioester exchange reactions (leading to native chemical ligation, NCL) to create magnetoresponsive materials, which potentially have applications in biomedicine. Firstly, iron oxide magnetic nanoparticles have been synthesised using a thermal co-precipitation method followed by chemical modification with sulfhydryl motifs for use as smart biomaterials. Knowing that the behaviour and reactivity of nanoparticles is highly influenced by their physicochemical properties, a thourough characterisation of these particles has been obtained. Secondly, during this project, several thioester derivatives have been synthesised that can be incorporated into the membranes of 800 nm liposomes. Among these, the spectrophotometric properties of synthetic lipid 38 allowed the investigation of trans-thioesterification rates with cysteinyl functionalities, both in solution and at the phospholipid membrane interface of liposomes. Product identification has been achieved using mass spectrometry and 1H-NMR spectroscopy. Finally, the conditions required to induce the release of a dye (e.g. 5(6)-CF) from MNP-V upon exposure to an AMF pulse have been established. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases has been investigated as interesting payload due to its fluorescent and anti-viral properties.

Hydrogels are popular materials for biological applications since they exhibit properties like that of natural soft tissue and have tunable properties. Biodegradable hydrogels provide an added advantage in that they degrade in an aqueous environment thereby avoiding the need for removal after the useful lifetime. In this work, we investigated poly(β-amino ester) (PBAE) biodegradable hydrogel systems. To begin, the factors affecting the macromer synthesis procedure were studied to optimize the reproducibility of the resulting hydrogels made and create new methods of tuning the properties. Hydrogel behavior was then tuned by altering the hydrophilic/hydrophobic balance of the chemicals used in the synthesis to develop systems with linear and two-phase degradation profiles. The goal of the research was to better understand methods of controlling hydrogel properties to develop systems for several biomedical applications. Several systems with a range of properties were synthesized, and their in vitro behavior was characterized (degradation, mechanical properties, cellular response, etc.). From these studies, materials were chosen to serve as porogen materials and an outer matrix material to create a composite scaffold for tissue engineering. In most cases, a porous three dimensional scaffold is ideal for cellular growth and infiltration. In this work, a composite with a slow degrading outer matrix PBAE with fast degrading PBAE microparticles was created. First, a procedure for developing porogen particles of controlled size from a fast-degrading hydrogel material was developed. Porogen particles were then entrapped in the outer hydrogel matrix during polymerization. The resulting composite systems were degraded and the viability of these systems as tissue engineering scaffolds was studied. In a second area of work, two polymer systems, one PBAE hydrogel and one sol-gel material were altered through the addition of iron oxide nanoparticles to create materials with remote controlled properties. Iron oxide nanoparticles have the ability to heat in an alternating magnetic field due to the relaxation processes. The incorporation of these nanoscale heating sources into thermosensitive polymer systems allowed remote actuation of the physical properties. These materials would be ideal for use in applications where the system can be changed externally such as in remote controlled drug delivery.

Nanostructured materials have attracted a broad interest for applications in scientific and engineering fields due to their extraordinary properties stemming from the nanoscale dimensions. This dissertation presents the development of nanomaterials used for different applications, namely biomedicine and dye lasing. Various inorganic nanoparticles have been developed as contrast agents for non-invasive medical imaging, such as magnetic resonance imaging (MRI) and X-ray computed tomography (CT), owing to their unique properties for efficient contrasting effect. Superparamagnetic iron oxide nanoparticles (SPIONs) are synthesized by thermo-decomposition method and phase-transferred to be hydrophilic used as MRI T2 (negative) contrast agents. Effects of surface modification of SPIONs by mesoporous silica (mSiO2) coating have been examined on the magnetic relaxivities. These contrast agents (Fe3O4@mSiO2) were found to have a coating-thickness dependent relaxation behavior and exhibit much higher contrast efficiency than that for the commercial ones. By growing thermo-sensitive poly(N-isopropylacrylamide -co-acrylamide) (P(NIPAAm-co-AAm)) as the outermost layer on Fe3O4@mSiO2 through free radical polymerization, a multifunctional core-shell nano-composite has been built up. Responding to the temperature change, these particles demonstrate phase transition behavior and were used for thermo-triggered magnetic separation. Their lower critical solution temperature (LCST) can be subtly tuned from ca. 34 to ca. 42 ˚C, suitable for further in vivo applications. An all-in-one contrast agent for MRI, CT and fluorescence imaging has been synthesized by depositing gadolinium oxide carbonate hydrate [Gd2O(CO3)2·H2O] shell on mSiO2-coated gold nanorod (Au NR), and then the particles were grafted with antibiofouling copolymer which can further link with the fluorescent dye. It shows both a higher CT and MRI contrast than the clinical iodine and gadolinium chelate contrast agent, respectively. Apart from the imaging application, owing to the morphology of Au NR, the particle has a plasmonic property of absorbing near-infrared (NIR) irradiation and suitable for future photothermal therapy. Cytotoxicity and biocompatibility of aforementioned nanoparticles have been evaluated and minor negative effects were found, which support their further development for medical applications. Gold nanoparticles embedded in the optical gain material, water solution of Rhodamine 6G (Rh6G) in particular, used in dye lasers can both increase and damp the dye fluorescence, thus, changing the laser output intensity. The studies of size effect and coating of gold nanoparticles on photostability of the gain media reveal that small sized (ca. 5.5 nm) gold nanoparticles are found detrimental to the photostability, while for the larger ones (ca. 25 nm) fluorescence enhancement rather than quenching is likely to occur. And a noticeable improvement of the photostability for the gain material is achieved when gold is coated with SiO2.
QC 20120605

The work reported in this dissertation describes the design and synthesis of different gold nanoshells with strong absorption coefficients at the near-infrared region (NIR) of the spectrum, and includes preliminary studies of their use for the photo-induced heating of pancreatic cancer cells and ex vivo tissues. As the emphasis was on gold nanoshells with maximum extinctions located at 800 nm, the methods explored for their synthesis led us to the preparation of silica-core and hollow gold nanoshells of improved stability, with maximum extinctions at or beyond the targeted within the near-infrared window. The synthesis of silica-core gold nanoshells was investigated first given its relevance as one of the pioneering methods to produce gold nanostructures with strong absorption and scattering coefficients in the visible and the near-infrared regions of the spectrum. By using a classical method of synthesis, we explored the aging of the precursor materials and the effect of using higher concentrations than the customary for the reduction of gold during the shell growth. We found that the aging for one week of the as-prepared or purified precursors, namely, the gold cluster suspensions, and the seeded silica particles, along with higher concentrations of gold in the plating solution, produced fully coated nanoshells of 120 nm in size with smooth surfaces and maximum extinctions around 800 nm. Additional work carried out to reduce the time and steps in the synthesis of silica-core gold nanoshells, led us to improve the seeding step by increasing the ionic strength of the cluster suspension, and also to explore the growth of gold on tin-seeded silica nanoparticles. The synthesis of hollow gold nanoshells (HGS) of with maximum extinctions at the NIR via the galvanic replacement of silver nanoparticles for gold in solution was explored next. A first method explored led us to obtain HGS with maximum extinctions between 650 and 800 nm and sizes between 30 and 80 nm from silver nanoparticles, which were grown by the addition of silver nitrate and a mild reducer. We developed a second method that led us to obtain HGS with maximum extinctions between 750 and 950 nm by adjusting the pH of the precursor solution of the silver particles without much effort or additional steps. The last part of this work consisted in demonstrating the photo-induced heating of two biological systems containing HGS. Photothermal therapy studies of immobilized PANC1 pancreas cancer cells in well-plates were carried out with functionalized HGS. We found that cells exposed to HGS remained viable after incubation. Moreover, the cells incubated with HGS modified with mercaptoundecanoic acid and folic acid turned non-viable after being irradiated with a laser at 800 nm. The other study consisted in the laser-induced heating between 750 and 1000 nm of ex vivo tissues of chicken and pork with nanoshells injected. In comparison with non-injected tissues, it was found that the temperature at the irradiated areas with HGS increased more than 10 °C. Moreover, the extent of the heated area was broader when the laser was used at wavelengths beyond 900 nm, suggesting that the heating was due to the radiation absorbed and transformed into heat primarily by the HGS and at a lesser extent by the water in the tissue.

The present work is divided into two sections. The first section deals with the synthesis of regenerable adsorbents for the removal of arsenic from contaminated waters. An adsorbent based on carboxymethylated polyethylenimine grafted agarose gels was synthesized and characterized as a regenerable synthetic ferric oxide adsorbent with high capacity for arsenate ions at pH 3.0. Similarly, four metal ion chelating adsorbents based on dipicolylamine were synthesized and characterized with respect to their Cu(II), Fe(III) and As(V) adsorption capacities. The most efficient adsorbents were Nov-PEI-DPA and Nov-TREN-DPA. Additionally, a commercial ion exchange resin was modified with permanganate to oxidize arsenite into arsenate. A complete oxidation-adsorption system was proposed in which a column packed with the oxidation resin was connected in series with an adsorbent column composed of the polyethylenimine grafted agarose gels.The second section involved work with magnetic nanoparticles. First, composite adsorbents consisting of magnetic particles encapsulated within agarose beads with and without grafted iminodiacetic acid (IDA) chelating groups were synthesized. The adsorption capacity of the adsorbents for Cu(II), Fe(III) and As(V) at different concentrations was investigated. Batch experiments were carried out to determine the Fe(III) and As(V) adsorption isotherms for the magnetic Novarose-IDA. Regenerability of the adsorbent was achieved with a pH change of the inlet solution, without affecting its magnetic or adsorption properties.Magnetic composite particles were synthesized for biomedical applications. First, magnetic nanoparticles were coated with silica and then used for gold nanoshell production. These nanoshells were functionalized with a Brij S10 derivative, containing carboxylic groups, using dodecanethiol as a bridging agent to incorporate a fluorescent biomolecule.Finally, magnetic and gold particles were encapsulated in PLGA nanoparticles. Docetaxel was loaded on these multifunctional nanoparticles and released studies were performed at 37°C. The presence of magnetite, colloidal gold and gold nanoshells in the PLGA nanoparticles was revealed by the coloration acquired by the polymeric nanoparticles. The release of drug from the polymeric nanoparticles showed a biphasic behavior with an initial burst followed by a prolonged slow release. There was no effect of the presence of magnetic or metallic particles on docetaxel release.

There is an undeniable link between oxidative stress, inflammation, and disease. Currently, approaches using antioxidant therapies have been largely unsuccessful due to poor delivery and bioavailability. Responding to these limitations, we have developed classes of polymer and delivery systems that can overcome the challenges of antioxidant and anti-inflammatory therapy. In our initial studies, nanoparticles of poly(trolox), a polymeric form of trolox, were surface-modified with antibodies. This modification allows for specific targeting to endothelial cells, affording controllable and localized protection against oxidative stress. We have shown these targeted nanoparticles bind, internalize, and provide protection against oxidative stress generation and cytotoxicity from iron oxide nanoparticles. In a similar fashion, we have tested the ability of poly(trolox) to prevent rheumatoid arthritis in vivo. Poly(trolox) nanoparticles were encapsulated in a PEGylated polymer to enhance circulation and biocompatibility. These particles were shown to accumulate in inflamed joint tissue, recover natural antioxidant function, suppress protein oxidation, and inhibit inflammatory markers. Lastly, we developed a class of polyphenolic compounds utilizing a non-free radical based reaction chemistry of poly(β-amino esters). The polyphenol apigenin was investigated for its anti-inflammatory properties to inhibit inflammation-mediated tumor cell metastasis. PEGylated nanoparticles that incorporated apigenin poly(β-amino ester) were developed and found to retain their anti-inflammatory efficacy while providing a long term release profile. These inhibited the ability of tumor cells to adhere to inflamed vascular cells. We also have shown that these polymers can suppress markers of inflammation responsible in enhancing tumor cell adhesion.

The optical and chemical properties of the materials used in catalytic and sensing applications directly determine the characteristics of the resultant catalyst or sensor. It is well known that a catalyst needs to have high activity, selectivity, and stability to be viable in an industrial setting. The hydrogenation activity of palladium catalysts is known to be excellent, but the industrial applications are limited by the cost of obtaining catalyst in amounts large enough to make their use economical. As a result, alloying palladium with a cheaper, more widely available metal while maintaining the high catalytic activity seen in monometallic catalysts is, therefore, an attractive option. Similarly, the optical properties of nanoscale materials used for sensing must be attuned to their application. By adjusting the shape and composition of nanoparticles used in such applications, very fine changes can be made to the frequency of light that they absorb most efficiently. The design, synthesis, and characterization of (i) size controlled monometallic palladium nanoparticles for catalytic applications, (ii) nickel-palladium bimetallic nanoparticles and (iii) silver-palladium nanoparticles with applications in drug detection and biosensing through surface plasmon resonance, respectively, will be discussed. The composition, size, and shape of the nanoparticles formed were controlled through the use of wet chemistry techniques. After synthesis, the nanoparticles were analyzed using physical and chemical characterization techniques such as X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Scanning Transmission Electron Microscopy- Energy-Dispersive Spectrometry (STEM-EDX). The Pd and Ni-Pd nanoparticles were then supported on silica for catalytic testing using mass spectrometry. The optical properties of the Ag-Pd nanoparticles in suspension were further investigated using ultraviolet-visible spectrometry (UV-Vis). Monometallic palladium particles have been synthesized and characterized to establish the effects of nanoparticle size on catalytic activity in methanol decomposition. The physicochemical properties of the synthesized palladium-nickel nanoparticles will be discussed, as a function of the synthesis parameters. The optical characteristics of the Ag and Pd nanoparticles will be determined, with a view toward tuning the response of the nanoparticles for incorporation in sensors. Analysis of the monometallic palladium particles revealed a dependence of syngas production on nanoparticle size. The peak and steady state TOFs increased roughly linearly with the average nanoparticle diameter. The amount of coke deposited on the particle surfaces was found to be independent on the size of the nanoparticles. Shape control of the nickel-palladium nanoparticles with a high selectivity for (100) and (110) facets (≤ 80%) has been demonstrated. The resulting alloy nanoparticles were found to have homogeneous composition throughout their volume and maintain FCC crystal structure. Substitution of Ni atoms in the Pd lattice at a 1:3 molar ratio was found to induce lattice strains of ~1%. The Ag nanocubes synthesized exhibited behavior very similar to literature values, when taken on their own, exhibiting a pair of distinct absorbance peaks at 350 nm and 455 nm. In physical mixtures with the Pd nanoparticles synthesized, their behavior showed that the peak position of the Ag nanocubes' absorbance in UV-Vis could be tuned based on the relative proportions of the Ag and Pd nanoparticles present in the suspension analysed. The Ag polyhedra synthesized for comparison showed a broad doublet peak throughout the majority of the visible range before testing as a component in a physical mixture with the Pd nanoparticles. The addition of Pd nanoparticles to form a physical mixture resulted in some damping of the doublet peak observed as well as a corresponding shift in the baseline absorbance proportional to the amount of Pd added to the mixture.

Currently, cancer is the second leading cause of death in the United States. Conventional cancer treatment includes chemotherapy, radiation, and surgical resection, but unfortunately, all of these methods have significant drawbacks. Hyperthermia, the heating of cancerous tissues to between 41 and 45°C, has been shown to improve the efficacy of cancer therapy when used in conjunction with irradiation and/or chemotherapy. In this work, a novel method for remotely administering heat is presented. This method involves heating of tumor tissue using hydrogel nanocomposites containing magnetic nanoparticles which can be remotely heated upon exposure to an external alternating magnetic field (AMF). The iron oxide nanoparticles contained in the hydrogel nanocomposites are able to heat via an AMF due to Brownian and Neel relaxation processes. The administration of hyperthermia via hydrogel nanocomposites allows for local delivery of heat to tumor tissue while also providing a drug depot to deliver chemotherapeutic agents. Both in vivo and in vitro studies have demonstrated that numerous chemotherapeutic agents, when used in conjunction with hyperthermia, show improved efficacy in treating cancer Various magnetic hydrogel nanocomposites were synthesized and characterized for this work including poly(ethylene glycol) (PEG)-based hydrogels, which were studied due to their inherent biocompatibility and “stealth” properties, as well as, poly(β-amino ester) (PBAE)-based hydrogels which have tailorable degradation properties. The PEG hydrogels were investigated for their temperature-responsiveness swelling, mechanical strength, heating capabilities, biocompatibility, ability to kill M059K glioblastoma cells via thermoablation, and the ability to deliver paclitaxel, a chemotherapeutic agent. PBAE hydrogels were also characterized for their degradation and swelling properties, ability to heat upon exposure to an AMF, biocompatibility, mechanical strength, and ability to deliver paclitaxel in a controlled fashion. Additionally, multiple cancer cell lines were exposed to a combination of paclitaxel and heat (at 42.5 °C) in vitro and it was shown that A539 lung carcinoma cells exhibit higher cytotoxicity when exposed to both heat and paclitaxel than either treatment alone. Overall, magnetic hydrogel nanocomposites are promising materials that can be utilized for the multi-modality treatment of cancer through the synergistic delivery of both heat and chemotherapeutic agents.

Iron oxide nanoparticles (IONPs) have been widely studied for a variety of applications, from biomedical applications (e.g., cell separation, drug delivery, contrast agent for magnetic resonance imaging and magnetically mediated energy delivery for cancer treatment) to environmental remediations (e.g., heavy metal removal and organic pollutants degradation). It has been demonstrated that IONPs can induce the production of reactive oxygen species (ROS) via Fenton/Haber-Weiss reactions which has been shown to be one of the key underlying mechanisms of nanoparticles toxicity. This inherent toxicity of nanoparticles has been shown to enhance the efficacy of traditional cancer therapies such as chemotherapy and radiation. In addition, the generation of ROS induced by IONPs has been also studied as advanced oxidation processes (AOP) for wastewater treatment. Recent research has also shown that exposure to an alternating magnetic field can significantly enhance the generation of ROS induced by IONPs. Moreover, the coatings of IONPs play an important role on the surface reactivity of nanoparticles since it can prevent the generation of ROS via Fenton chemistries at the surface of the nanoparticles. In this work, co-precipitated IONPs were functionalized with small molecules including citric acid, sodium phosphate, amino silane and dopamine. The impact of coating on surface reactivity of the as-synthesized particles was studied using methylene blue dye degradation assay under AMF exposure. With the coatings of these small molecules, the IONPs induced ROS generation was significantly decreased because of the dense surface coverage. To study the effect of polymeric coatings, a degradable poly (beta amino ester) (PBAE) polymer coating was synthesized with dopamine as an anchor to bind to nanoparticles. The surface reactivity of the particles was expected to be recovered once the polymer coating was degraded. Furthermore, the impact of non-degradable PEG-based polymer coating on surface reactivity via ROS generation was also investigated using methylene blue decolorization assay with the presence of AMF. The retention of surface reactivity of PEG-based polymer coated IONPs shows promise for cancer treatment. The application of IONPs as heterogeneous catalyst for organic contaminant degradation was investigated. Bisphenol A (BPA) was used as a model compound, and Fenton reactions were induced by IONPs with the presence of hydrogen peroxide and hydroxylamine as well as alternating magnetic field exposure. The kinetics of BPA degradation under water bath and AMF exposure at 37oC was also studied, and the results showed potential applications of IONPs for organic pollutants remediation.

The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.

Les travaux réalisés aux cours de cette thèse ont portés sur la création de nouveaux complexes borates fluorescents. Des voies de synthèse relativement simples et efficaces ont permis d'accéder à deux nouvelles familles de fluorophores : les Boranils et les Boricos. Les Boranils présentent des coefficients d'absorption molaire élevés, des rendements quantiques pouvant atteindre 90% et la capacité à agir comme antenne efficace pour du transfert d'énergie photoinduit. De nombreuses modifications post-synthétiques ont été mises au point permettant l'accès à des fonctions de greffage utile pour des applications dans le domaine des cristaux liquides ou l'imagerie biomédicale. Enfin, l'extension de la conjugaison des Boranils a permis de déplacer les émissions vers le proche infrarouge. Les Boricos présentent des coefficients d'absorption élevés, des rendements quantiques allant jusqu'à 81% et la capacité à agir comme antenne ou accepteur dans des systèmes de transfert d'énergie photoinduit.

La technique de nanoprécipitation est une méthode simple et reproductible pour la synthèse de nanocapsules à coeur huileux recouvertes d’une enveloppe de polymères hydrophiles réticulés (polysaccharides, glycopolymères vinyliques…) en une seule étape. Grâce à leur biocompatibilité, leur biodégradabilité et leur activité biologique adaptables, les protéines constituent une autre grande famille de biopolymères d’intérêt pour des applications dans le domaine de l’encapsulation. Cependant, la production de nanocapsules protéiques par nanoprécipitation n’a jamais été décrite. Dans ce contexte, l’objectif principal de ce travail de thèse a été l’évaluation du potentiel d’une famille de protéines, les Suckerines, pour le procédé de nanoprécipitation. Les Suckerines sont une famille de protéines issues des dents décorant les ventouses du calamar géant Humboldt avec de prometteuses applications dans le domaine biomédical. Ces protéines possèdent une structure modulaire de type copolymère à bloc capable de former des feuillets bêta conférant de bonnes propriétés mécaniques. Les suckerines étant solubles dans une solution tampon composée d’acide acétique (pH 3) mais fortement agrégées dans les conditions de pH (valeurs comprises entre 5 et 10) classiquement utilisées pour la préparation de nanocapsules à coeur huileux par basculement de solvant, nous avons finalement choisi d’explorer la nanoprécipitation des protéines par salt shifting et donc la préparation de nanoparticules protéiques. L’utilisation du persulfate d’ammonium comme agent de coacervation et précurseur de radicaux et du tris(2,2′ bipyridyl)dichlororuthenium(II) hexahydrate a permis de produire des nanoparticules de suckerine de tailles modulables (100-185 nm de diamètre). Ces nanoparticules présentent des structures secondaires type feuillets bêta qui sont à l’origine du module de Young très élevé observé pour ces nano-objets (de l’ordre de grandeur du GPa). Une protéine de fusion, soluble en milieu aqueux à pH 7 a spécialement été conçue par voie de recombinaison dans le but de générer des nanocapsules protéiques par nanoprécipitation. Cette protéine (suckerine-soie) est formée d’un bloc central de peptide dérivé de suckerine de calamar promouvant une stabilité structurelle et deux blocs terminaux issus de fibroïnes de soie qui permettent à la protéine de fusion d’être soluble à un pH physiologique. Ce design moléculaire a permis la fabrication de nanocapsules remplies respectivement de hexadécane ou de miglyol avec une enveloppe de suckerine-soie et de tailles de l’ordre de grandeur de 190 à 250 nm. Finalement, aspirant à encapsuler un principe actif anti-cancéreux dans les nanocapsules à base de glycogène, nous avons développé un protocole où la méthode de nanoprécipitation est utilisée pour produire des nanoparticules de prodrogue entourés de glycogène
The nanoprecipitation technique is a reliable route to synthesize oil filled nanocapsules with shells made of hydrophilic polymers such as polysaccharides and vinyl based glycopolymers in a one pot procedure. Thanks to their biocompatibility, biodegradability and tunable biological activity, proteins are another promising class of materials for encapsulation purposes. However, the generation of proteinaceous nanocapsules by nanoprecipitation has never been reported. In this context, the main objective of this PhD was to evaluate the potential of a family of proteins, the Suckerins, in nanoprecipitation processes. Suckerins are a family of proteins found in the sucker ring teeth of the giant Humboltd squid with promising biomedical applications. These proteins possess a modular, block copolymer like structure capable of forming β-sheets responsible for good mechanical properties. The suckerin proteins are not soluble at a pH range between 5 and 10, a requirement of the nanoprecipitation technique. However, they can be solubilized using aqueous buffers at pH 3 containing acetic acid. Other ways of precipitating the protein were explored in this manuscript with salt shifting using ammonium persulphate as coacervation agents being capable of generating 100 nm nanoparticles. These nanoparticles presented the β sheet secondary structure which resulted in Young modulus in the GPa range. A fusion protein that could be solubilized in aqueous solutions at pH 7, and therefore be used in the nanoprecipitration process, was recombinantly produced. The protein (suckerin silk) is formed by a central squid suckerin-derived peptide block that provides structural stability and both termini from silk fibroins that make the modular protein highly soluble at physiological pH. This molecular design allowed the fabrication of hexadecane and miglyol filled nanocapsules with suckerin silk shells and sizes in the range 190 – 250 nm. Finally, aiming to encapsulate an anti cancer drug in glycogen nanocapsules we developed a protocol where the nanoprecipitation process is used to generate glycogen coated prodrug nanoparticles

Modern day medicine is on the brink of a new age of therapy, which aims to harness the natural power of molecular biology for disease treatment. This therapy could include replacement of dysfunctional genes that cause disorders such as cystic fibrosis (Lommatzsch and Aris, 2009), or silencing the overexpression of genes that cause disorders such as cancer (Pelengaris and Khan, 2003). In both examples, the treatment of these genetic diseases lies in the delivery of synthetic nucleic acids into diseased cells, the former being called gene replacement therapy (Dobson, 2006a), and the latter being called RNA interference (RNAi) therapy (Whitehead et al., 2009). While these techniques have long been in use as genetic research tools for gene transfection or silencing in vitro, their translation for use in clinical disease treatment has yet to be achieved. The main problem facing the development of these novel therapies is the specific delivery of nucleic acids into diseased cells within the body. It is hoped that nanoparticles (NPs) can be used to overcome this problem, by acting as vehicles to transport nucleic acids through the body for specific delivery into diseased cells. This feat can be aided by the attachment of additional functional molecules such as cell penetrating peptides (CPPs), targeting peptides, additional drug types and molecules for imaging during treatment. Many different NP design strategies are currently under development. It is essential for new designs to be extensively tested for toxicity and efficiency in human cells before they can be successfully released into the clinic. As part of this effort, this PhD project has investigated two different NP design strategies for drug delivery: 1) the use of a magnetic field (MF) and a CPP to increase the delivery of iron oxide magnetic NPs (mNPs) to cells grown in tissueequivalent 3D collagen gels, and 2) gold NPs (AuNPs) for the delivery of siRNA to silence the c-myc oncogene for cancer treatment. In the first investigation, a MF and the CPP penetratin were found to increase mNP delivery to cells grown in 3D. In the second investigation, AuNPs were assessed in a range of different cell types (grown in 2D) for their performance in 4 main areas; cellular toxicity, cellular uptake, c-myc knockdown and effect on the cell cycle.

Surface topography is known to be important in biomedical applications such as scaffolds for tissue regeneration and has been shown to affect wettability and cell behaviour. Traditionally, topographical effects such as surface texturing have been generated using methods such as photolithography, soft lithography, thermal embossing, and laser/electron beam techniques. This thesis introduces a relatively new technique known as photoembossing to create surface texturing for biomedical applications. Photoembossing is used to produce surface texturing on polymer surfaces by patterned ultraviolet (UV) exposure of a photopolymer blend without an etching step or an expensive mould. After a short general introduction and a literature review, the first experimental chapters describe surface patterning of poly(methyl methacrylate) (PMMA) photopolymer substrates by photoembossing. PMMA is blended with an acrylate monomer and photoinitiator by dissolution in a volatile solvent and processed into films by wire bar coating, and fibres are produced by electrospinning. Surface texture is achieved on both films and fibres by photoembossing. Endothelial cell culture shows that the substrates are biocompatible and cells readily adhere to the surface. In tissue regeneration applications, scaffold degradation is often important to allow tissue in-growth. Thus, in subsequent studies polylactide-co-glycolide (PLGA) is used as a polymer binder. PLGA blended with a triacrylate monomer showed partial degradation after 10 weeks, with a cross-linked acrylate network remaining. Endothelial cell adhesion was even better on the PLGA photopolymer substrates compared to PMMA. Furthermore, surface texture improved cell adhesion and proliferation on the PLGA photopolymer. To obtain completely degradable substrates, thiol monomer was used in addition to the acrylate to produce ester bonds after the thiol-ene reaction, which is cleavable by hydrolysis. Accelerated degradation in sodium hydroxide (NaOH) showed complete degradation of this photopolymer system. The degradation rate of the photopolymer could be tuned by the molecular weight of the acrylate monomer, with low molecular weight monomers degrading more slowly than high molecular weight species. Furthermore, the height of the surface relief structures could be enhanced by using low-molecular-weight acrylate monomers. Endothelial cell culture revealed biocompatibility of the blend and cells were able to adhere after 24 hours of seeding. This thesis demonstrates that photoembossing is a viable technique in producing surface texture for tissue engineering applications. This surface texture can be achieved on both biocompatible and biodegradable photopolymer films and fibres.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

In the past few years, significant progress in the study of scaffolds for cells grow has taken place. This research has led to the development of a wide variety of metallic, polymeric, ceramic and composite biomaterials. This thesis describes the development of a novel composite system with tunable morphological and mechanical properties, ease of production and capability to guide the biological response. The composite system was composed by polyamide 6 (PA6) and carboxyl-functionalized multi-walled carbon nanotubes (MWCNT), which were used as reinforcement agents in the polymer matrix. Electrospinning was used as the fabrication technique for the production of anisotropic networks. Physical and biological properties of the nets were evaluated focusing on the effect of the filler addition. It was observed that the production technique induced the alignment of MWCNT within the nanofiber axis and the formation of a roughness on the fiber's surface. The biological properties of MG63 and MRC5 cell lines were enhanced if compared with the neat PA6 networks due to surface modification caused by the filler addition.

In the past few years, significant progress in the study of scaffolds for cells grow has taken place. This research has led to the development of a wide variety of metallic, polymeric, ceramic and composite biomaterials. This thesis describes the development of a novel composite system with tunable morphological and mechanical properties, ease of production and capability to guide the biological response. The composite system was composed by polyamide 6 (PA6) and carboxyl-functionalized multi-walled carbon nanotubes (MWCNT), which were used as reinforcement agents in the polymer matrix. Electrospinning was used as the fabrication technique for the production of anisotropic networks. Physical and biological properties of the nets were evaluated focusing on the effect of the filler addition. It was observed that the production technique induced the alignment of MWCNT within the nanofiber axis and the formation of a roughness on the fiber's surface. The biological properties of MG63 and MRC5 cell lines were enhanced if compared with the neat PA6 networks due to surface modification caused by the filler addition.