表面等離子共振 (SPR) 是受激發的電子的總體振動，一般在金屬、電介質界面上發生。我們能以振盪的電場去激發SPR。由於表面等離子共振時會產生巨大的增強電場，這令他在近場光學與納米光學中有著廣泛的應用。例如：微流控芯片、等離子波導、隱形裝置等等。 / 在這論文中，我們會研究納米金屬體之間的作用力。基於以下原因，這在納米技術中是一個重要課題。第一，當了解到物體之間的作用力後，我們可以以此開發出把納米尺寸的物體移動與放置的方法，有助於用自下而上式的方法製作納米器件。第二，物體之間的作用力會改變器件中納米顆粒的位置，因而會影響器件的特性。 / 在一般情況下，納米尺寸的物體的作用力都可以略去不理的，因為作用力是與體積成正比。但是，當表面等離子共振發生時，相互作用力會急速地增強。這增強是由於金屬顆粒的電偶會急速地增強的原故。我們稱這現象為「表面等離子共振增強光泳」 (SPREP). / 這論文由三個主要部份組成。第一部份，我們研究一帶梯度的金屬納米球與一振盪及旋進電偶之間的相互作用。我們以第一原理進路去解決這問題，並作了長波長假設。我們的解析解能夠處理多極效應，這效應在外場不均勻時是不可忽略的。我們探討了作用力、力矩、電場分佈。更發現了，當金屬納米球的梯度很高時，電偶與金屬納米球之間與有一穩定的力平衡。這研究有助於開發新型的光學鑷子。 / 第二部份，我們探討兩個金屬納米球之間的 SPREP，我們介紹了不同的計算方法。Bergman-Milton譜表示以及多重鏡象法。 兩個金屬納米球之間也有著穩定的平衡， 這表示在一群納米球中，可能有著穩定結構。這穩定的平衡，是由於表面等離子共振的頻率與相互距離有關，這是一種多體效應。這研究有助於了解納米簇的結構形成。 / 最後，我們以離散偶極子近似法(DDA)研究多體問題，雖然DDA並不是精確解，但當顆粒之間相距不太接近時，這依然是一個良好的近似。當顆粒的數量太多時，我們以等效介質理論去著手，不再考慮每一顆粒各自的位置，而只考慮顆粒的濃度。 / Surface plasmon resonance (SPR) is the collective electrons excitations, which occurred at the metal-dielectric interfaces and can be induced by an oscillating electric field. Because of the large field enhancement, SPR has a wide range of applications in near field optics and nano-optics, such as biosensors, lab-ona- chip devices, plasmonic waveguides, and cloaking devices. / In this thesis, we study the interparticle forces between metallic nanosized objects. It is an important topic in nanotechnology for at least two reasons. Firstly, the study of the interparticle forces may provide methods to control the motion and position of nano-size objects, which can be used to fabricate artificial nano-structure by bottom up approach. Secondly, the force can change the arrangement of the particles in the nanodevices and hence affecting the property of the devices. / The interparticle forces of nano-sized dielectric particles are negligible, since the force is proportional to the volume of the objects. However, the interparticle forces of metallic particles will be greatly enhanced when SPR occurs, which is able to compensate the volume effect. This phenomenon is called surface plasmon resonance enhanced photophoresis (SPREP), which is one of the consequences of the rapid increase in the dipole moment in the particles. / This thesis is consisted of three main parts. In the first part, we study the SPREP between a graded metallic nanosphere and a point dipole which is undergo oscillation and precession. A first principle approach is applied to handle this problem. Our analytic solutions are able to capture the multipole effect, which cannot be neglected in highly non-uniform fields. We have analyzed three important physical quantities: the induced force, the induced torque, and the field distribution. Furthermore, we find that there is a binding between the nanoparticle and the dipole source, when the gradation of the graded particles is large enough. This study has a potential application in developing a novel kind optical tweezers. / In the second part, we study the SPREP between two metallic nanoparticles. The force spectra are calculated by two different methods: Bergman- Milton spectral representation and multiple image method. The binding between two nanoparticles is observed, which indicates a possible stable structure among the metallic clusters. The binding is caused by the excitation of collective plasmon modes, and the consequence that the resonance poles sℓ are the functions of separation distances. This study may provide a better understanding in the structure formation of colloidal clusters in nano-scales. / Finally, we consider a many-particle system by the discrete dipole approximation (DDA) and effective medium theory. Although, the DDA is not an exact formalism, it is a suitable approximation for considering finite number of particles, if the distances among particles are not too close. When the number of particles in the host solution is large, we can use the effective medium theory to handle the problem. Instead of considering all discrete particles individually, we will consider the interaction between a single particle and a new effective host solution, where the dielectric function of the effective host solution is determined by the concentration of nanoparticles in the host solution. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Kin Lok = 納米金屬顆粒中的表面等離子共振增強光泳 / 陳建樂. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 90-94). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chan, Kin Lok = Na mi jin shu ke li zhong de biao mian deng li zi gong zhen zeng qiang guang yong / Chen Jianle. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Nanoparticles and nanotechnology --- p.1 / Chapter 1.2 --- The history of nanoparticles and nano-optics --- p.1 / Chapter 1.3 --- Applications of nanoparticles --- p.2 / Chapter 1.3.1 --- Optical applications --- p.2 / Chapter 1.3.2 --- Biological and medical applications --- p.3 / Chapter 1.4 --- Electrokinetics of nanoparticles --- p.4 / Chapter 1.4.1 --- Review on recent work on electrokinetics --- p.5 / Chapter 1.5 --- Objectives of the thesis --- p.6 / Chapter 2 --- Basic Principles --- p.8 / Chapter 2.1 --- Drude model --- p.8 / Chapter 2.2 --- Complex dielectric function --- p.9 / Chapter 2.2.1 --- Electric field in an imperfect conductor --- p.10 / Chapter 2.3 --- Effective medium theory --- p.11 / Chapter 2.3.1 --- Maxwell-Garnett approximation --- p.12 / Chapter 2.3.2 --- Bruggeman approximation --- p.13 / Chapter 2.3.3 --- Bergman-Milton spectral representation (BMSR) --- p.13 / Chapter 2.3.4 --- Effective dielectric function of shelled sphere --- p.17 / Chapter 2.4 --- Surface plasmon resonance (SPR) --- p.18 / Chapter 2.5 --- Surface plasmon resonance enhanced photophoresis (SPREP) --- p.20 / Chapter 2.6 --- Justification of long wavelength limit --- p.23 / Chapter 3 --- Manipulation of Nanoparticles by a Single Dipole Source --- p.25 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Formalism --- p.26 / Chapter 3.2.1 --- Electrostatic potential of a dipole --- p.27 / Chapter 3.2.2 --- Electrostatic potential of a dipole in terms of multipole expansion --- p.27 / Chapter 3.2.3 --- Laplace's equation of graded sphere --- p.30 / Chapter 3.2.4 --- Boundary value problem --- p.31 / Chapter 3.2.5 --- Force --- p.33 / Chapter 3.2.6 --- Torque --- p.35 / Chapter 3.3 --- Result and discussion --- p.36 / Chapter 3.3.1 --- Force --- p.38 / Chapter 3.3.2 --- Torque --- p.45 / Chapter 3.3.3 --- Electric field distribution --- p.46 / Chapter 3.4 --- Conclusion --- p.48 / Chapter 4 --- Interaction between Two Objects --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Interaction between two particles --- p.50 / Chapter 4.2.1 --- Dipole approximation --- p.50 / Chapter 4.2.2 --- Multiple images method --- p.52 / Chapter 4.2.3 --- Bergman-Milton spectral representation for collection of grains --- p.58 / Chapter 4.2.4 --- Equation of motion --- p.61 / Chapter 4.2.5 --- Result and discussion --- p.62 / Chapter 4.3 --- Particle near a conducting plane --- p.67 / Chapter 4.3.1 --- Dipole approximation --- p.67 / Chapter 4.3.2 --- Multiple image method --- p.69 / Chapter 4.3.3 --- Result and discussion --- p.70 / Chapter 5 --- Many-particle Systems --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Discrete dipole approximation --- p.72 / Chapter 5.2.1 --- 2-particle system --- p.73 / Chapter 5.2.2 --- 4-particle system --- p.74 / Chapter 5.2.3 --- Result and discussion --- p.75 / Chapter 6 --- Concentration Effect --- p.80 / Chapter 6.1 --- Introduction --- p.80 / Chapter 6.2 --- Formalism --- p.81 / Chapter 6.2.1 --- Result and discussion --- p.83 / Chapter 7 --- Summary --- p.88 / Bibliography --- p.90 / Chapter A --- Eigenfunctions, Eigenvalues, and Green's function --- p.95 / Chapter A.1 --- Isolated sphere --- p.95 / Chapter A.1.1 --- Eigenfunctions and eigenvalues --- p.96 / Chapter A.1.2 --- Green's function --- p.98 / Chapter A.2 --- Planar interface --- p.98 / Chapter A.2.1 --- Eigenfunctions and eigenvalues --- p.99 / Chapter A.2.2 --- Green's function --- p.100 / Chapter B --- Property of Spherical Harmonics and Associated Legendre Polynomials --- p.101 / Chapter B.1 --- Complex conjugate of Yℓm(Ω): --- p.102 / Chapter B.2 --- Differential Property --- p.102 / Chapter B.3 --- Limiting value --- p.102

Supported lipid bilayers (SLBs) are one of the most common model membranes used in the field of cell membrane biology as they provide a well-defined model membrane platform for determination of molecular-level interactions between different biomolecules (e.g. proteins, peptides) and lipid membrane. Compared to model organisms, the use of SLB is preferable since it mimics cell plasma membrane in a very simple and well-controlled way. Therefore, molecular structure of membrane and experimental conditions (e.g. solution chemistry, temperature, and pH) can be easily adjusted to the required conditions of any systematic research. In addition, SLBs are typically easy to form, cheap and very reproducible and they are compatible with different surface characterization techniques, such as quartz crystal microbalance with dissipation (QCM-D), ellipsometry and atomic force microscopy (AFM). This study demonstrates that QCM-D analysis of SLBs serve as powerful tool to investigate and characterize the mechanisms of interactions between lipid membrane and gold nanoparticles (NPs), environmentally relevant polymers, and disease-inducing peptides. Due to many critical applications of gold NPs in drug delivery and diagnostics, understanding of membrane-NP interactions is crucial especially for determination of NPs cytotoxicity. In this study we focus on membrane disruption as one of the different mechanisms by which metal NPs induce cytotoxicity. The use of SLB is beneficial for this goal as it elucidates the unique mechanism of membrane disruption without interference of other mechanisms taking place simultaneously in biological cells. For NP-membrane interaction studies, a SLB composed of L-α-phosphatidylcholine (egg PC) was formed on a SiO2-coated crystal and QCM-D analysis was performed to obtain information about mass and viscoelastic changes of SLB resulting from interactions with gold NPs. For better understanding of the mechanisms of NP-membrane interactions, we systematically changed the NPs properties and the experimental conditions. In order to understand the effect of NP size, gold NPs with diameters of 2,5,10, and 40 nm were tested and compared to each other. NPs were tested in their citric acid-stabilized state as well as in the presence of poly (methacrylic acid) (PMAA), representing an organic coating that could become associated with NPs in the environment. The results indicated that when dissolved in water, gold NPs with the dimeters of 2, 5, 10, and 40 nm did not perturb the membrane, but in the presence of environmentally relevant polymer, the larger nanoparticles were found to disrupt the membrane. In order to elucidate the effect of surface chemistry, 10 nm - gold NPs with various functionalizations (i.e. anionic, cationic and non-ionic ligands) were tested. Control experiments were designed to test the effect of NPs in the absence of humic substances which means the NPs were dissolved in water. In these cases, regardless of the type of NP functionalization, no substantial bilayer mass changes were observed. This suggests that the charge and chemistry of the ligands had a minor effect on NP-membrane interactions. Furthermore, in both the control and humic acid experiments, there were small dissipation changes (less than 1 unit) indicating that the overall membrane structure was not perturbed. In order to mimic environmentally-relevant conditions, mass and viscoelasticity of SLB was characterized in the presence of four different natural polymers, also known as natural organic materials (NOMs): Fulvic and humic acids extracted from Suwannee River (SRFA and SRHA), which had relatively lower molecular weights and a commercial humic acid (HA) and the humic acid extracted from Elliott soil (ESHA) with higher molecular weight. The results showed that NOMs with lower molecular weights, adsorbed to the bilayer, while higher molecular weight components, did not induce any changes to the bilayers. In addition, the NPs in SRFA and SRHA increased the mass of the bilayer by 20-30 ng, while the NPs in HA and ESHA changed the mass of the bilayer by < 10 ng. It was concluded that the presence of humic substances as well as their physical and chemical properties exert a direct impact on the interactions between cell membrane and the nanoparticles. In addition to the field of NP toxicity, SLBs play a pivotal role in the field of neurodegenerative diseases, such as Alzheimer’s disease (AD), in which the pathological cascade of events starts from interactions of a misfolded peptide with cell membrane. In this thesis, we confirm the validity of QCM-D analysis of SLB as an important platform for investigation of amyloid β (the peptide associated with AD) interactions with lipid membrane. Adsorption of Aβ peptide to cell membrane is known to take place on the so-called “lipid raftâ€� which are membrane microdomains enriched with cholesterol, sphingomyelin and ganglioside. The formation of SLBs containing lipid rafts is not only important for the field of AD research, but also it is important for other in vitro studies of cell biology as the lipid rafts are responsible for a variety of biological functions such as association of some membrane proteins and cellular signaling. However, the presence of lipid raft components such as sphingomyelin and cholesterol makes the formation of the bilayer more challenging which leads to adsorption of intact vesicles on the substrate without formation of the bilayer. In this study, the formation of lipid bilayer composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), cholesterol (Chol), sphingomyelin (SM), and ganglioside (GM) was investigated using QCM-D. A challenge was that the raft-containing vesicles remained intact on the SiO2 crystal. Therefore, different experimental conditions were tested to induce vesicle fusion, such as pH, temperature, osmotic pressure, and vesicle size. The key parameter in forming the bilayer was found to be applying osmotic pressure to the vesicles by having the vesicles exterior concentration of NaCl higher than interior concentration. When this concentration gradient was applied to the vesicles before flowing them on the substrate, vesicle rupture was favored and formation of a complete bilayer could occur. Here, we report the effects of each tested variable on the adsorption and fusion of the raft-containing vesicles, and the results are discussed based on the mechanisms of vesicle-vesicle and vesicle-substrate interactions.After developing the robust method for formation of SLB with lipid rafts, we used that as a template to characterize the mechanism of interactions between Aβ peptide and cell membrane which leads to onset of AD. The mechanism of Aβ toxicity leading to AD has not fully discovered yet, due to the complexity of the process including several steps of Aβ peptide adsorption on membrane, conformational change from disordered in solution to a membrane-bound α-helix structure and then formation of β-sheet aggregates that serve as fibrillation seeds. In this study, we showed that QCM-D technique as a promising tool to conduct systematic studies on the mechanism of interactions between Aβ peptide with lipid membrane. To our knowledge, this was the first time QCM-D was utilized for characterization of Aβ fibrillation starting from monomer states until formation of mature fibrils. The data indicated that peptide-membrane interactions follow a two-step kinetic pathway starting with the adsorption of small (low-n) oligomers until covering all the adsorption sites on the surface. In the second step, the membrane structure is destabilized as the result of interaction with oligomers which leads to lipid loss from the surface. Consistency of the results with the data obtained via other techniques substantiates QCM-D technique as a robust approach to answer the remaining unanswered questions in the field of Alzheimer’s disease.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-07-24T14:58:19Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Mariane A Sanson.pdf: 3058378 bytes, checksum: 43bb48ea0be645db69444ec684b43bac (MD5) / Made available in DSpace on 2018-07-24T14:58:19Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Mariane A Sanson.pdf: 3058378 bytes, checksum: 43bb48ea0be645db69444ec684b43bac (MD5) Previous issue date: 2016-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Compostos de ouro já apresentam propriedades anti-inflamatórias estabelecidas pela literatura, além de importante potencial antioxidante. A utilização do ouro na forma nanoparticulada é uma aposta promissora para o tratamento de condições inflamatórias, como a doença periodontal, pelo seu mecanismo de ação. Apesar da biocompatibilidade estabelecida do ouro, é importante atentar-se para a possibilidade de efeito tóxico do ouro na forma nanoparticulada, devido a sua maior facilidade de penetrar nos tecidos e atravessar membranas, em resposta principalmente ao seu tamanho e concentração de ouro presente. O objetivo deste estudo foi a caracterização das nanopartículas de ouro (AuNPs) de 10, 20 e 30 nm utilizadas no trabalho, bem como testar seu efeito tópico em modelos de inflamação aguda local in vivo (edema de orelha induzido por TPA e edema de pata induzido por LPS) e verificar sua citotoxicidade em cultura de fibroblastos (3T3) e macrófagos (RAW 264.7) murinos após 12, 24 e 48h pelos ensaios de MTT, VN e teste de exclusão do azul de tripan. Para a caracterização das AuNPs foram realizados os testes de espectroscopia no ultravioleta-visível (Uv-Vis), difratometria de raios-x, absorbância atômica, potencial zeta e microscopia eletrônica de varredura por canhão de emissão de campo elétrico (FEG). Os resultados dos testes de caracterização demonstraram que as nanopartículas apresentavam-se estáveis e realmente com os diâmetros de 10, 20 e 30 nm, como proposto pelo método de síntese, estando adequadas para a utilização nos testes subsequentes. No modelo de edema de orelha induzido por TPA as formulações de AuNPs em creme a 0,1% reduziu a formação do edema em 47,76%, enquanto a formulação em creme a 0,06% reduziu o edema em 33,72% após 6h. Após 24h todas as formulações testadas apresentaram redução da formação do edema, apresentando resultados sem diferença estatística em comparação ao grupo tratado com a dexametasona. No modelo de edema de pata induzido por LPS a formulação de AuNPs em creme a 0,1% foi capaz de reduzir a formação do edema em 84,46% e a formulação em creme a 0,6% reduziu em 86,57%, também sem apresentar diferença estatística em comparação ao grupo tratado com dexametasona. Quanto aos testes de viabilidade celular, os métodos do MTT e VN não foram capazes de obter resultados confiáveis, porém o teste de exclusão do azul de tripan indicou ausência de citotoxicidade das AuNPs em 12h nas culturas de fibroblastos e macrófagos. Após 24h de incubação, a maior concentração (35 mg/L) das dispersões de AuNPs nos 3 diâmetros testados reduziu a viabilidade de macrófagos, e os fibroblastos obtiveram seus valores de viabilidade celular diminuídos pela maioria das dispersões de AuNPs testadas. Após 48h de incubação, todas as concentrações e diâmetros testados das dispersões de AuNPs foram capazes de diminuir drasticamente as porcentagens de viabilidade celular tanto em fibroblastos quanto em macrófagos. / Gold compounds already demonstrated anti-inflammatory properties in previous studies, in addition to important antioxidant potential. The use of gold in nanoparticle form is promising for the treatment of inflammatory conditions such as periodontal disease, especially for its mechanism of action. Although the established gold biocompatibility, it is important to pay attention to the possible toxic effect of gold on nanoparticulate form, for its ease of penetration through the tissues and membranes, mainly in response to its size and gold concentration. The study purpose was the physical characterization of gold nanoparticles (AuNPs) of 10, 20 and 30 nm used in the experiments, in addition to test their topical effect in models of acute inflammation in vivo (ear edema TPA-induced and paw edema LPS-induced) and verify its cytotoxicity in fibroblasts and macrophages mice cell cultures after 12, 24 e 48 hours by MTT and Neutral Red assay and exclusion of trypan blue test. To characterize the AuNPs were performed the ultraviolet-visible spectroscopy, x-ray diffraction, atomic absorbance, zeta potential and Field Emission Gun Scanning Electron Microscopy (FEG). The characterization tests results shown that the nanoparticles are stable and truly presented the diameters of 10, 20 and 30 nm, as proposed by synthesis method, and so are suitable for use in subsequent experiments. In the ear edema TPA-induced model AuNPs cream formulation at 0.1% reduced edema formation at 47.76%, while the cream formulation at 0.06% reduced 33.72% of edema after 6 hours. After 24 hours all formulations tested showed a reduction of edema formation, presenting results with no statistical difference compared to the group treated with dexamethasone. In LPS-induced paw edema model the AuNPs cream formulation at 0.1% was able to reduce edema formation in 84.46% and cream formulation at 0.6% decreased 86.57%, also without showing statistical difference compared to the group treated with dexamethasone. In viability tests, the MTT and Neutral Red methods were not able to obtain reliable results, but exclusion trypan blue test indicated no cytotoxicity of AuNPs after 12h in fibroblasts and macrophages cell cultures. After 24h incubation, the dispersions of AuNPs in 3 diameters at the highest concentration (35 mg/L) reduced the macrophages viability and fibroblast viability decreased with most of the tested AuNPs dispersions. After 48h all tested AuNPs dispersions concentrations and diameters were able to reduce the percentages of fibroblasts and macrophages cell viability.

Membrane proteins are important targets that represent more than 50% of current drug targets. However, characterisation of membrane proteins falls behind compared to their soluble counterparts. The most challenging part of membrane protein research is finding a suitable membrane mimetic that stabilises them in solution and maintains their native structure and function. The recently developed saposin-A (SapA) based lipid nanoparticle system seems to be advantageous over existing membrane mimetic system. It provides a native-like lipid bilayer, high incorporation yield and more importantly size adaptability. SapA lipid nanoparticles have been applied to structural studies and two high-resolution structures of membrane proteins were previously obtained using cryo-electron microscopy. This thesis aimed to study small-to-medium sized membrane proteins in SapA lipid nanoparticles using NMR spectroscopy. We first explore the mechanism of SapA lipid nanoparticle formation for the purpose of establishing an incorporation protocol that can be applied to most membrane proteins. The effect of pH and the presence of detergents on the opening of SapA was investigated in Chapter 2. A proposed energy diagram describing the mechanism of SapA opening is reported with which we were able to develop a protocol that can generate different sizes of SapA-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) nanoparticles. In addition, we also showed that SapA can form lipid nanoparticles with various lipid compositions, showing the versatility of the system. In Chapter 3, we validated the ability of SapA lipid nanoparticles to be used as a membrane mimetic. A -barrel model protein, bacterial outer membrane protein X (OmpX), was incorporated into SapA-DMPC nanoparticles and a 2D 15N-1H correlation NMR spectrum was recorded. Our result was compared to the NMR parameters of the same protein in MSP nanodiscs from the literature, and it was concluded that SapA lipid nanoparticles indeed provide a lipid bilayer environment similar to MSP nanodiscs. Because of high incorporation yield, we were able to incorporate OmpX into different lipid compositions to investigate the effect of lipid head groups and aliphatic chains on the membrane protein's chemical environment. Next, the applicability of SapA lipid nanoparticles was expanded to -helical transmembrane proteins in Chapter 4. Two microbial rhodopsins, Anabaena sensory rhodopsin (ASR) and Natronomonas pharaonis sensory rhodopsin II (pSRII), were tested. The parameters for expression and purification of ASR were first screened for the optimal yield. Although incorporation of ASR resulted in inhomogeneous particles due to imperfect experimental procedure, pSRII in SapA-DMPC nanoparticles showed high sample quality. The 2D NMR spectrum of pSRII in SapA-DMPC nanoparticles shows distinct differences to pSRII in detergent micelles, suggesting substantial effects from the membrane mimetic on the conformation of the membrane protein. Despite the good NMR spectral quality considering the large particle size, perdeuteration of pSRII and the lipids will be necessary for further investigation. With the SapA lipid nanoparticles established, we aimed to use it for the study of a biologically important G protein-coupled receptor, 1-adrenergic receptor (1AR), discussed in Chapter 5. The possibility of expressing 1AR using a cell-free expression system was explored first. Although a good amount of the protein was obtained, only a fraction of it was functional. Therefore, a conventional baculovirus-insect cell expression system was used to produce selective isotope labelled 1AR for NMR studies. NMR spectra of 1AR in SapA-DMPC nanoparticles with activating ligands and an intracellular binding partner were recorded and compared to the spectra of the same protein in detergents. This revealed a more active-like conformation of ligand-bound 1AR in the lipid bilayer, suggesting that certain parts of the protein are sensitive to the membrane mimetic used. This emphasises the importance of using a native-like membrane mimetic to capture the full properties of membrane proteins. In conclusion, I demonstrate in this thesis that SapA lipid nanoparticles are a versatile membrane mimetic system that can accommodate membrane proteins with different sizes and folds. This system is also compatible with solution NMR spectroscopy enabling structure and dynamics studies of biologically important membrane proteins. We believe SapA lipid nanoparticles will have a significant impact on membrane protein research in the future.

Orientador: Sidney José Lima Ribeiro / Banca: Younes Messaddeq / Banca: Danilo Manzani / Resumo: Este trabalho descreve o preparo de bioconjugados obtidos pela associação das proteínas fluorescentes Azurite, EGFP, mTangerine e mCherry com nanopartículas de ortovanadato de ítrio dopadas com európio (YVO4:Eu3+) para o estudo de processos de transferência de energia que viabilizem o aumento da emissão do íon Eu3+. A reação em estado sólido do carbonato de sódio com o precursor metavanadato de sódio foi utilizada para a obtenção do ortovanadato de sódio, o qual foi empregado na síntese das nanopartículas pelo método de coprecipitação. As nanopartículas obtidas apresentaram diâmetro médio de 44 nm e foram funcionalizadas com o grupo maleimida pela reação do citrato com a 2-maleimidoetilamina através do acoplamento carbodiimida EDC/Sulfo-NHS para posterior bioconjugação com as proteínas fluorescentes. Sob excitação na região do UV, as nanopartículas apresentaram intensa emissão na região vermelha, característica da transição 5D0→7F2 do Eu3+ em 618 nm. Plasmídeos contendo os genes de codificação das proteínas Azurite e mTangerine foram construídos, subclonados em vetores pQE-9 e geneticamente modificados para expressão em linhagens de E. coli apropriadas. As proteínas EGFP e mCherry também foram expressas a partir de plasmídeos previamente preparados, seguidas de purificação por cromatografia de afinidade juntamente com as proteínas Azurite e mTangerine. O rendimento médio de expressão obtido foi de 90 mg L-1 e as 4 proteínas expressas foram bioconjugadas às nanopartículas de E... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work describes the preparation of bioconjugates through the combination of Azurite, EGFP, mTangerine and mCherry fluorescent proteins with europium doped yttrium orthovanadate nanoparticles (YVO4:Eu3+) for energy transfer studies aiming the sensitization of Eu3+ emission. The solid state reaction between sodium carbonate and sodium metavanadate was performed to obtain the sodium orthovanadate, which was used for the nanoparticles synthesis by the co-precipitation method. The obtained nanoparticles present mean diameter of 44 nm and were functionalized with maleimide group through carbodiimide EDC/Sulfo-NHS coupling for further bioconjugation with the fluorescent proteins. Under UV excitation, the nanoparticles displayed strong red emission, characteristic of the 5D0→7F2 Eu3+ transition at 618 nm. Plasmids containing the Azurite and mTangerine genes were developed, cloned into pQE-9 vector and mutated for expression in E. coli strains. The EGFP and mCherry proteins were also expressed from previously prepared plasmids, followed by affinity chromatography purification along with the Azurite and mTangerine proteins. The mean expression yield was 90 mg L-1 and all proteins were conjugated with the nanoparticles through thiol-maleimide click chemistry. The results showed that under 307 nm excitation, Azurite and EGFP emission energy is non-radiatively transferred to Eu3+ 5D0 emitting level, increasing both emission lifetime and intensity of the nanoparticles. The bioconjugati... (Complete abstract click electronic access below) / Mestre

Il existe plusieurs types d’agressions environnementales : biologiques (virus, bactéries…), chimiques (gaz, fumées, métaux…), physiques (bruits, rayonnements…), et d’autres telles que le stress par exemple. L’appareil respiratoire, qui représente une interface majeure avec l’environnement, est particulièrement vulnérable vis-à-vis de ces agressions, qui ont souvent des conséquences pulmonaires, pouvant parfois conduire au décès. Le tabac notamment est la cause de près de 100 millions de décès au cours du XXème siècle d’après l’Organisation Mondiale de la Santé (OMS), et sera la cause d’environ un milliard de décès au prochain siècle. L’exposition à la fumée de cigarette engendre une inflammation chronique et est souvent corrélée au développement de cancers (1), mais induit aussi de nombreuses autres pathologies pulmonaires telles que la broncho-pneumopathie chronique obstructive (BPCO) / There are several types of environmental attacks: biological (viruses, bacteria …), chemical (gases, smokes, metals …), physical appearances (rumours, brilliances …), and others such as the stress for example. The respiratory system, which represents a major interface with the environment, is particularly vulnerable towards these attacks, which often have lung consequences, being able to sometimes lead to the death. The tobacco in particular is the cause of about 100 million deaths during the XXth century according to the World Health Organization (WHO), and will be the cause about a billion deaths in the next century. The exhibition in the smoke of cigarette engenders a chronic inflammation and is often correlated in the development of cancers (1), but also leads of numerous

Doctor of Philosophy / Department of Chemistry / Santosh Aryal / Nanotechnology offers exciting options for the site-selective delivery of chemotherapeutics and diagnostic agents using nanoparticles. Varieties of organic and inorganic nanomaterials have been explored extensively as a delivery system either in the form of drug carriers or imaging agents. Successful stories include the clinical translation of anticancer nanomedicines such as PEGylated liposomal doxorubicin (DOXIL®), albumin-bound paclitaxel (Abraxane®), and polymeric micelle loaded paclitaxel (Genexol®), which are currently used in the clinic as one of the first lines for cancer chemotherapies. These conventional nanomedicines rely on passive-drug targeting taking advantage of leaky tumor vasculature, called the Enhanced Permeability and Retention (EPR) effect. However, delivering biologically active components selectively to the diseased cell, for example, cancer, is highly challenging due to the biological barriers in the body including blood pool cells/proteins, heterogeneous microenvironment, and intracellular degradation. Therefore, the goal of this dissertation is to develop nanoplatforms that can deliver the agents of interest in targeted fashion to cancer while bypassing or collaborating with the biological barriers. The design consideration of these nanoplatforms centralizes on using simple chemical reactions and cell biology to engineer nanoparticles. The presented nanoparticles were extensively studied and evaluated for their biological functions using in vitro and in vivo models. These nanoconstructs described herein address current limitations of conventional nanomedicine such as (1) the lack of understanding of the interaction of nanoparticle and biological system, and (2) the lack of an effective targeting strategy to deliver drugs to the cancer cell in the tumors. The significant findings of each system will be highlighted and discussed throughout this dissertation. Results obtained highlight key findings such as NP intracellular fate, maximized tumor accumulation, and unique pharmacokinetics could open the avenues for systemic investigations for personalized medicine and lay the foundation for nanomedicine design to accelerate clinical translation.

O estudo de nanopartículas plasmônicas de ouro sob o ponto de vista conceitual foi o foco desta tese, explorando principalmente os efeitos do envoltório molecular e a intensificação dos espectros SERS tendo em vista aplicações em sensoriamento químico. Como moléculas sonda, foram selecionadas espécies multifuncionais, como a 2,4,6-trimercapto-1,3,5-triazina (TMT), 4,5-diamino-2,6- dimercaptopirimidina (DadMcP ou Dad) e a mercaptoetilpirazina (PZT), que apresentam grupos tióis capazes de ancorar nas nanopartículas de ouro, deixando outros sítios livres para interagir com substratos e complexos metálicos. Observou-se que o envoltório molecular formado no método de Turkevich, é bastante dependente das condições de síntese, tendo sido possível detectar a presença do intermediário da reação de oxidação do citrato na superfície das nanopartículas, sob condições controladas, influenciando drasticamente o comportamento SERS. Foi feito um estudo sistemático da molécula sonda 2,4,6-trimercapto-1,3,5-triazina ancorada nas nanopartículas de ouro, tanto por troca da camada passivante (citrato), como por síntese in situ com e sem agente redutor. Esses sistemas foram investigados, sob diferentes condições, como sensores SERS para metais. Esse estudo foi ampliado para a molécula sonda 4,5-diamino-2,6-dimercaptopirimidina (DadMcP), explorando a influência do tempo na coordenação dessa espécie na superfície e o efeito de diferentes eletrólitos nos processos de agregação. Finalmente, foram apresentadas fortes evidências da ocorrência de processos fotoinduzidos envolvendo as nanopartículas funcionalizadas, com destaque para a mercaptoetilpirazina (PZT), cujo comportamento mostrou-se bastante inusitado, gerando filmes fotoagregados sob influência da luz UV com possível aplicação em fotolitografia. / The study of plasmonic gold nanoparticles under the conceptual point of view was the focus of this thesis, exploring mainly, the effects of molecular shell and the intensification of SERS spectra aiming at applications in chemical sensing. For the probe molecules, multifunctional species were selected, such as a 2,4,6-trimercapto- 1,3,5-triazine (TMT), 4,5-diamine-2,6-dimercaptopyrimidine (DadMcP or Dad) and mercaptoethylpyrazine (PZT) which present thiol groups able to anchor onto gold nanoparticles, leaving available sites for further interaction with substrates and metal complexes. It was observed that the molecular shell in Turkevich\'s method is very dependent on the synthesis condition, being possible to detect the intermediate product of citrate oxidation reaction in the nanoparticle surface, under controlled conditions, dramatically influencing the SERS behavior. A systematic study was conduct with the probe molecule 2,4,6-trimercapto-1,3,5-triazine anchored to gold nanoparticles either by changing the passivating layer (citrate), or for in situ synthesis with and without a reducing agent. These systems were investigated under different conditions as SERS sensors for metals. This study was extended to the probe molecule 4,5-diamine-2,6-dimercaptopyrimidine, exploiting the influence of time in the coordination of such species and also the effect of different kinds of eletrolytes in the aggregation process. Finally, it has been presented strong evidences for the occurence of photoinduced processes involving functionalized nanoparticles with emphasis on mercaptoethylpyrazine, whose bahavior has proved to be very unusual, generating photoaggregated films under UV light influence, with possible applications in photolithography.

Polymer based membranes play a key role in several industrially important gas separation technologies, e.g., removing CO2 from natural gas, with enormous economic and environmental impact. In this thesis, we develop a novel hybrid membrane construct comprised entirely of inorganic nanoparticles grafted with polymer chains. For all graft architectures studied, the permeability of several small gases and condensable solvents are higher in GNP membranes than the neat polymer analogs. More interestingly, the matrix-free GNPs displayed a non-monotonic peak in gas permeability as a function of grafted chain molecular weight, M_n, at a fixed grafting density, σ. Furthermore, in contrast to neat polymer membranes, which suffer from degraded performance over time due to chain densification and “aging”, the performance of GNP membranes is preserved for months to years. We show that these enhancements are not limited to a single polymer, thus we suggest that this grafting mechanism may be an option to improve permeability in polymer membranes in general. We conjecture the grafted polymer chains must stretch to fill the interstitial voids in the NP “lattice”, as such voids would be free-energetically unfavorable due to the relatively high surface tension of the polymer melt. Since this stretching leads to an unfavorable chain conformational entropy, we expect a decrease in the polymer density, which we verify experimentally as well as through molecular dynamics simulations. When a penetrant molecule is placed in these regions of highest distortion, the chains can assume more favored, undistorted conformations. This in turn creates a driving force for further penetrant uptake. Therefore, we systematically study the structure and dynamics of matrix-free GNP materials at various chain grafting densities and a wide range of graft molecular weight. Small angle scattering experiments reveal that the core nanoparticle spacing systematically increases with increasing molecular weight but the overall morphology remains amorphous and isotropic. Whereas previous studies1 have found the brush height in matrix-free GNPs scales as the degree of polymerization 〖~N〗^0.5, we find that the brush height in our systems scales 〖~N〗^0.7, indicating the chains are indeed highly stretched. Moreover, studies of the structural evolution upon swelling with solvent show that the brush is fully wetted and the solvent distribution is homogeneous within the film. Additionally, we systematically probe the dynamics of matrix-free GNP systems over broad length and time scales using linear and non-linear mechanical rheology, and broadband dielectric spectroscopy. The linear viscoelastic response shows that while the polymeric signal (e.g. glassy and Rouse dynamics) is equivalent for a range of graft chain lengths, the terminal flow of these materials is slowed by several decades compared to the neat melts of corresponding molecular weight. The low frequency (long time) response shows that below a critical molecular weight, these systems transition from polymeric to that of a colloidal system. To understand this behavior, a scaling theory is developed to describe the polymer brush conformation, which reveals that at this transition point the grafted particles behave as a system of packed “rigid” spheres. We note that the transition point coincides with the maximum observed in the transport behavior, and that the reduced system mobility may be responsible for the reduced aging effects. On the other hand, secondary relaxations for GNPs at this transition molecular weight are found to be faster than the neat polymer of corresponding molecular weight, which is attributed to a lower effective polymer density found in these samples. Therefore, the critical question underpinning this work is: how do the structure and dynamics influence and/or result from increased free volume in matrix-free grafted nanoparticle materials? We conclude that matrix-free grafted nanoparticle constructs allow for precise control of structure-property relationships over multiple length scales, and serve as a novel materials design platform with the potential to function as high performance gas separation technologies.

Ce travail décrit la séparation de microphase à l'état solide d'un copolymère dibloc comprenant un bloc hautement biocompatible poly [2 - (méthacryloyloxy) éthyl phosphorylcholine] et un bloc pH-sensible poly [2 - (diisopropylamino) méthacrylate d'éthyle] (PMPC-b-PDPA). L'auto-assemblage d'un copolymère dibloc amphiphiles à base de polystyrène (PS) et le poly (acide acrylique) (PAA), a été étudié en suspension aqueuse, et leur décoration par du chitosane, afin de vérifier leur capacité à encapsuler et à libérer par voie transdermique la finastéride, une molécule stéroïdiens inhibiteur de la enzyme 5-alpha-réductase, qui a été recommandée pour le traitement de l'alopécie androgénétique. La morphologie des films PMPC30-b-PDPA60 a été analysée par SAXS et (S) TEM. Films du PMPC30-b-PDPA60 préparés dans une solution de l'éthanol à température ambiante présentent une morphologie cylindrique, qui subit une transition ordre-ordre sur un recuit thermique à 170 ° C: la structure lamellaire résultant coexiste avec une proportion de cylindres organisée dans une phase hexagonale compacte. En revanche, les films du copolymère préparé à partir de méthanol ne subissent pas la même transition morphologique, résultant dans des structures mal organisées, indépendante de traitement thermique. Enfin, les structures lamellaires sont obtenues directement à partir d'une solution aqueuse à pH 4, sans traitement thermique. Ces systèmes offrent une nouvelle alternative pour la fabrication de structures lamellaires constituées d'un matériau biomimétique et anti-fouling, élargissant l'éventail des possibilités dans le domaine de l'ingénierie macromoléculaire.Une autre stratégie adoptée dans ce travail a été basé sur le développement de nanoparticules bien organisé avec des propriétés de surface nature bioinspirés, formé entre polymersomes chargés négativement à base des copolymères à blocs de polystyrène (PS) et le poly (acide acrylique) (PAA) décorés avec du chitosane, un polysaccharide de charge opposée. Le rôle de l'adsorption du chitosane avec deux poids moléculaires distincts (chitosane oligosaccharides et du chitosane avec un faible poids moléculaire) sur la surface des nanoparticules PS139-b-PAA17 et des nanoparticules PS404-b-PAA63 ont été démontrés par diffusion dynamique de la lumière, potentiel zêta et caractérisation morphologiques. En présence du chitosane, le potentiel zêta de polymersomes devient positif. Ce résultat a été interprété en termes d'interactions électrostatiques, qui induisent une adsorption du chitosane sur la surface des polymersomes. Ce résultat a été confirmé par une observation comparative par microscopie entre des polymersomes et des polymersomes décorées. Polymersomes avec un diamètre <200 nm et une distribution granulométrique relativement étroite ont été obtenus pour les deux systèmes. L'effet de la décoration des nanoparticules par du chitosane sur la perméation cutanée in vitro du finastéride, incorporé dans la paroi hydrophobe de polymersomes, a également été évaluée. La pénétration cutanée du finastéride a été estimée par les paramètres de perméabilité tels que le flux, temps de latence et du coefficient de perméabilité de la finastéride. Une amélioration de la perméation du finastéride à partir des nanoparticules a été observée, en particulier à partir de nanoparticules décorées avec du chitosane. Le polymersome PS404-b-PAA63 décorées avec du chitosane semble être le système le plus approprié car il favorisé une meilleure rétention du médicament dans la peau et les faibles valeurs de flux de perméation, suggérant que le système fournit un véhicule de remplacement pour l'administration transdermique de finastéride. / This work describes the microphase separation in bulk of an diblock copolymer comprising a highly biocompatible poly[2-(methacryloyloxy)ethyl phosphorylcholine] block and a pH-sensitive poly[2-(diisopropylamino) ethyl methacrylate] block (PMPC-b-PDPA). The self-assembly of an amphiphilic diblock copolymer based on polystyrene (PS) and poly(acrylic acid) (PAA) was studied in terms of their decoration with the chitosan, verifying their ability to incorporate and transdermally release the drug finasteride, a steroidal molecule 5-alpha-reductase inhibitor that has been recommended for the treatment of androgenetic alopecia. The morphology of PMPC30-b-PDPA60 films was analyzed using SAXS and (S)TEM. PMPC30-b-PDPA60 films cast from ethanol solution at room temperature exhibit a thermodynamically quasi-stable cylindrical morphology, which undergoes an order-order transition upon thermal annealing at 170 oC: the resulting lamellar structure coexists with a minor proportion of cylinders organized into a hexagonal compact phase. In contrast, copolymer films cast from methanol do not undergo the same morphological transition. Instead, short-range liquid-like structures are obtained regardless of the annealing processes. Finally, direct self-assembly to form a lamellar morphology at room temperature can be achieved by solvent-casting from aqueous solution at pH 4. These systems offer a new alternative for the fabrication of lamellar structures in which one layer is biomimetic and non-fouling, expanding the range of possibilities in the macromolecular engineering field. Another strategy adopted in this work was based on the development of well-organized nanoparticles with nature-bioinspired surface properties, formed between negatively charged polymersomes based on polystyrene (PS) and poly(acrylic acid) (PAA) block copolymers decorated with chitosan, an oppositely charged polysaccharide. The role of chitosan with two distinct molecular weights (chitosan oligosaccharide and low molecular weight chitosan) adsorption on the surface of oppositely charged PS139-b-PAA17 and PS404-b-PAA63 nanoparticles were demonstrated by dynamic light scattering measurements, zeta potential and morphological characterization. In the presence of chitosan, the zeta potential of polymersomes becomes positive. This result was interpreted in terms of electrostatic interactions, which induce a flat adsorption of the chitosan on the surface of the polymersomes. This result was further confirmed by a comparative observation by microscopy of bare and chitosan-decorated polymersomes. Polymersomes with a diameter < 200 nm and a relatively narrow size distribution were obtained for both systems. The effect of chitosan decoration of self-assembled nanoparticles on skin penetration in vitro of finasteride was also evaluated, once incorporated in the wall hydrophobic of polymersomes. The skin permeation through pig ear skin of finasteride was estimated by the permeability parameters such as flux, lag time and permeability coefficient of finasteride. An improved permeation of finasteride from the nanoparticle system was observed, especially from nanoparticles decorated with chitosan. The PS404-b-PAA63 polymersome decorated with chitosan seems to be the most appropriate system since it provided higher drug retention in skin and low permeation flux values, suggesting that the PS-b-PAA/chitosan system provides an alternative for transdermal drug delivery system of finasteride.