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Haghighat, Manesh Mohamad Javad Haghighat. "Effects of the Nanoparticle Protein Corona on Nanoparticle-Cell Membrane Interactions." Ohio University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1597967288027448.
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Lucon, Janice Elizabeth. "Development of protein nanoparticle based composite materials." Diss., Montana State University, 2013. http://etd.lib.montana.edu/etd/2013/lucon/LuconJ0513.pdf.
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Inspired by the core-shell composite structures found in nature, a range of protein based composites have been developed. These materials were made using synthetic approaches, which utilized the native protein architecture as an initiation point and size constrained reaction vessel for the piecewise formation of the second material. In the first illustration of this approach, a protein-P t composite was formed, where the protein cage has been modified to include a metal binding moiety for improved synthesis of metallic P t nanoclusters, which were shown to be an active H ₂ catalyst. This composite was analyzed by native mass spectrometry to determine the number of P t ions bound prior to mineralization and to measure the distribution of species after mineralization, which provided a unique view into the mineralization process. The second illustration was a material synthesized using the cage-like protein architecture as an internal guiding synthetic scaffold for the formation of a coordination polymer core inside the protein cage. The construction of this coordination polymer was unusual in that unlike normal coordination polymer synthesis, coordination of the metal preceded formation the ditopic ligands, which were afterwards completed using azide-alkyne click chemistry. Finally, a collection of protein-polymer composites were developed, which utilized a living radical polymerization method, atom transfer radical polymerization, to form internal polymer cores. By labeling one of these protein-polymer constructs with a Gd based MRI contrast agent a material with vastly improved relaxivity was made. The development of each of these three types of composites served to improve our understanding of the natural systems, from which they are derived, and provide a basis for further development of advanced multicomponent nanomaterials. 'Co-authored by Md Joynal Abedin, Masaki Uchida, Lars Liepold, Craig C. Jolley, Mark Young, Trevor Douglas, Shefah Qazi, Gregory J. Bedwell, Ben LaFrance, and Peter E. Prevelige, Jr.'
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Pham, Tuan Anh [Verfasser]. "Protein assisted nanoparticle assembly and protein-nanocomposite fabrication / Tuan Anh Pham." Konstanz : Bibliothek der Universität Konstanz, 2016. http://d-nb.info/1119707870/34.
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Gallagher, Jane. "Protein nanoparticle conjugates for use in bioanalytical applications." Thesis, University of Strathclyde, 2011. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=17065.
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Maury, Pascale Anne. "Fabrication of nanoparticle and protein nanostructures using nanoimprint lithography." Enschede : University of Twente [Host], 2007. http://doc.utwente.nl/57701.
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Wright, Kimberley Elizabeth. "Engineering of a specific binding site for protein labelling with luminescent lanthanide coated nanoparticles : a study of protein labelling and nanoparticle-peptide interactions." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5576/.
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The work presented in this thesis investigates the use of new luminescent lanthanide complexes, both free and bound to the surface of gold nanoparticles, for protein labelling. Lanthanide complexes were shown to maintain their luminescence properties when conjugated to proteins and one complex also demonstrated participation in Förster resonance energy transfer when conjugated to a protein in an appropriate system. Furthermore, it was found that bovine serum albumin can act as a vehicle to transport luminescent lanthanide complexes into two human cell lines. Lanthanide complexes were then used to coat 13 nm gold nanoparticles for protein labelling within cells. The aim was to find a peptide sequence to preferentially bind to gold nanoparticles which could be expressed as part of a protein of interest, acting as a binding site within the cell. The interaction of peptides with gold nanoparticles was examined using several methods and, of the sequences tested, CCPGCC was found to have the highest affinity for the nanoparticles. This peptide was expressed in HeLa cells as part of green fluorescent protein. Co-localisation of the nanoparticles with the protein in cells could not be established through fluorescence microscopy, however, cell lysis revealed green fluorescence protein associated with nanoparticle aggregate.
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Fernandez, Maxence. "Auto-assemblage de nanoparticules métalliques et semi-conductrices dirigé par reconnaissance entre protéines artificielles." Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1S129.
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L’auto-assemblage de nanoparticules dirigé par des biomolécules constitue une approche prometteuse pour la mise au point de nanomatériaux structurés présentant des propriétés optiques collectives originales. L’objet de cette thèse concerne l’auto-assemblage de nanoparticules métalliques et semi-conductrices dirigé par des protéines artificielles appelées α-Repeat. Dans cette optique, des nanocristaux semi-conducteurs (CdSe/ZnS ou CdSe/CdS) et des nanoparticules d’or sphériques ou anisotropes ont été préparés. Ces nanoparticules ont été fonctionnalisées avec des ligands peptidiques PEGylés, qui leur confère une stabilité colloïdale satisfaisante tout en conservant leurs propriétés optiques. Une stratégie de fonctionnalisation basée sur des étiquettes d’affinité poly-cystéine et poly-histidine a permis de greffer les protéines sur la surface des nanoparticules inorganiques. Les nanoparticules ainsi fonctionnalisées avec les protéines artificielles ont ensuite été utilisées pour l’auto-assemblage de nanoparticules semi-conductrices et l’auto-assemblage hybride entre des nanoparticules semi-conductrices et des nanoparticules métalliques. L’étude structurale des ensembles obtenus a montré, dans certains cas, une interdistance bien définie et inférieure à 10 nm. Finalement, l’étude des propriétés optiques a révélé des transferts d’énergie non radiatifs entre nanoparticules semi-conductrices et nanoparticules métalliques, qui témoignent d’interactions exciton—plasmon très fortes induites par l’auto-assemblageNanoparticles self-assembly driven by biomolecules is a promising approach for developing nanostructured materials with new optical properties. The purpose of this work is the self-assembly of metal and semiconductor nanoparticles directed by artificial proteins called α-Repeat. For this purpose, semiconductor nanocrystals (CdSe/ZnS or CdSe/CdS) and spherical or anisotropic gold nanoparticles have been prepared. These nanoparticles have been functionalized with PEGylated peptide ligands providing them adequate colloidal stability while maintaining their optical properties. A functionalization strategy based on polycysteine and poly-histidine tags has allowed the proteins to be grafted onto the surface of inorganic nanoparticles. Nanoparticles functionalized with artificial proteins were then used for the self-assembly of semiconductor nanoparticles and hybrid self-assembly between semiconductor nanoparticles and metal nanoparticles. The structure study of self-assembled nanostructures has shown, in some cases, a very well defined sub-10 nm interparticle distance. Finally, the study of optical properties revealed very strong exciton-plasmon interactions induced by self-assembly. This self-assembling process strongly affected the emission properties of the semiconductor nanoparticles in hybrid ensembles
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Davidson, Patricia Marie L. "Langmuir films and nanoparticle applications of a spider silk protein analog." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100794.
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A synthetic analog of a spider silk protein (M4) was studied. Langmuir films were made and an inflexion in the isotherm indicated conformational changes upon compression. Deposition onto solid substrates was most successful using a hydrophobic substrate and the Langmuir-Schaeffer method. AFM was used to image the surface, which was mesh like and did not show any indication of order.Gold nanoparticles were produced in the presence of the protein and protein solutions were added to read made nanoparticles for the purpose of displacing the weak ligands present. CD measurements were performed on the protein solutions to study its conformation. Nanoparticle size information was obtained from TEM images. DLS was used to determine if the protein was affected by the addition of the gold nanoparticles. Precipitation of the protein was shown not to affect the nanoparticles.
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Zhang, Xiaolu. "NANOPARTICLE BEHAVIOR IN BIOLOGICAL GELS AND BIOFLUIDS: THE IMPACT OF INTERACTIONS WITH CHARGED BIOGELS AND THE FORMATION OF PROTEIN CORONAS ON NANOPARTICLES." UKnowledge, 2015. http://uknowledge.uky.edu/chemistry_etds/57.
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With the rapid growth of nanotechnology, situations where nanomaterials will interact with biological systems will unquestionably grow. Therefore, it is increasingly understood that interactions between nanomaterials and biological environments will play an essential role in nanomedicine. Biological polymer networks, including mucus and the extracellular matrix, serve as a filter for the exchange of molecules and nanoparticles. Such polymer networks are complex and heterogeneous hydrogel environments that regulate transport processes through finely tuned particle-network interactions. In chapters 3 and 4, we investigate the role of electrostatics on the basic mechanisms governing the diffusion of charged molecules inside model polymer networks by using fluorescence correlation spectroscopy (FCS). In chapter 3, we show that particle transport of charged probe molecules in charged hydrogels is highly asymmetric and that the filtering capability of the gel is sensitive to the solution ionic strength. Brownian dynamics simulations are in quantitative agreement with our experimental result. In chapter 4, we focus on hyperbranched cationic dendrimer macromolecules (polyamidoamine, PAMAM) which differ from probes in size, charge density and chain flexibilities. Our results show PAMAM has strongly reduced mobility in like charge gels and greatly enhanced apparent diffusivity in oppositely charged gels. Further studies with salt suggest that the oppositely charged polymer network acts as a giant counterion enhancing the mobility of PAMAM by changing its conformation to a more compacted state.
Due to their large surface areas, nanomaterials in biological fluids are modified by adsorption of biomolecules, mainly proteins, to form so called “protein coronas”. These coronas ultimately define the biological identity of the nanoparticles and dictate the interactions of cells with the protein-NP complex. We have studied the adsorption of human transferrin and bovine serum albumin on the surface of sulfonated polystyrene nanoparticle. In chapter 5, we show the formation of multi-layered protein coronas and compare to established adsorption models. In addition we followed for the first time the protein binding kinetics as a function of pH and salt. Through these studies, we aim to gain quantitative knowledge of the dynamic rearrangement of proteins on engineered nanomaterials.
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Giraudon--Colas, Gaël. "Caractérisation multiéchelle d'assemblages d'hémoglobine : de l'adsorption sur les nanoparticules aux gels nanocomposites Protein−Nanoparticle Interactions: What Are the Protein−Corona Thickness and Organization? In Situ Analysis of Weakly Bound Proteins Reveals Molecular Basis of Soft Corona Formation." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASF011.
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Les gels de protéine nanocomposites sont un sujet encore peu développé dans la littérature malgré de nombreuses applications allant de l’immobilisation d’enzyme aux prothèses en passant par les gels alimentaires. La protéine permet d’assurer la biocompatibilité des gels tandis que l’ajout des nanoparticules a pour but de moduler les propriétés mécaniques des gels. Nous avons donc décidé de nous intéresser aux gels d’hémoglobine réticulée chimiquement et dopés aux nanoparticules. L’hémoglobine (Hb) a été choisie pour sa grande abondance et ses propriétés de fixation du dioxygène. Les gels seront obtenus par réticulation par le glutaraldéhyde (GTA), un dialdéhyde très réactif. Les gels seront dopés par des nanoparticules de silice (NP) afin de comprendre déjà l’effet sur le gel du dopage par des nanoparticules modèles. La première partie de la thèse portera sur l’adsorption de l’hémoglobine sur les nanoparticules de silice afin de lever les dernières inconnues sur ce phénomène déjà étudié. Il sera mesuré les isothermes d’adsorption ainsi que l’activité de l’hémoglobine adsorbée. Les structures de l’hème, de la globine et de l’assemblage Hb/NP seront étudiées avec détails. Par la suite, les études se porteront sur les gels sans et avec nanoparticules afin d’élucider les effets de la gélification et du dopage respectivement. On déterminera les concentrations en Hb, GTA et NP permettant d’obtenir un gel. Puis, comme pour les assemblages Hb/NP, nous nous intéresserons à l’activité et à la structure de Hb (hème et globine). La structuration du gel sera de plus étudiée. Des études sur les propriétés élastiques des gels seront aussi menées et nous finirons sur la dynamique de la protéine gélifiée. Quand il sera possible, l’effet des concentrations des différents composants sera déterminé. Pour toutes ces études, il a été utilisé un vaste panel de techniques de caractérisation classique des protéines ou des gels. Beaucoup d’expériences ont été effectuées sur grands instruments (diffusion de rayonnement, spectroscopie d’adsorption X, dichroïsme circulaire). Des techniques plus accessibles comme la résonance paramagnétique électronique, la rhéologie ou la microscopie électronique ont aussi été employées. Les aspects les plus novateurs de cette thèse ont été l’effet de l’adsorption sur l’hème et la compréhension de la structure de la protéine gélifiée, deux aspects qui n’avaient pas été traitésNanocomposite protein gels are still an underdeveloped subject in the literature despite many applications ranging from enzyme immobilization to prostheses to food gels. The protein ensures the gel biocompatibility while the addition of the nanoparticles will modulate the gel mechanical properties. We decided to focus on chemically cross-linked hemoglobin gels doped with nanoparticles. Hemoglobin (Hb) was chosen for its high abundance and its oxygen binding properties. The gels will be obtained by crosslinking with glutaraldehyde (GTA), a very reactive dialdehyde. The gels will be doped with silica nanoparticles (NP) in order to understand the effect of doping with model nanoparticles on the gel. The first part of the work will focus on the hemoglobin adsorption on silica nanoparticles in order to resolve the remaining unknowns on this phenomenon, which has already been studied. The adsorption isotherms as well as the activity of the adsorbed hemoglobin will be measured. The structures of the heme, globin and the Hb/NP assembly will be studied in details. Subsequently, works will focus on gels without and with nanoparticles in order to respectively elucidate the effects of gelation and doping. We will determine the concentrations of Hb, GTA and NP to obtain a gel. Then, as with the Hb/NP assemblies, we will look at the activity and structure of Hb (heme and globin).The structuring of the gel will also be studied. Works on the gel elastic properties will also be carried out and we will finish on the dynamics of the gelled protein. When possible, the concentration effect for the different components will be determined. For all these studies, a large panel of conventional technics to characterize proteins or gels was used. Many experiments have been performed in synchrotrons and neutron research centers (radiation scattering, X-ray absorption spectroscopy, circular dichroism). Electronic paramagnetic resonance, rheology or electron microscopy, which are more accessible technics have also been employed. The most innovative aspects of this work were the effect of adsorption on heme and the understanding of the gelled protein structure, two aspects that had not been addressed until now
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Marichal, Laurent. "Interactions protéines-nanoparticules : émergence de nouveaux facteurs déterminant la formation de la couronne de protéines." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS100/document.
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Les nanoparticules sont de plus en plus présentes dans notre quotidien et leur présence dans les organismes vivants est aujourd’hui avérée. Aussi, dans un milieu biologique, des protéines recouvrent spontanément la surface des nanoparticules pour former une couronne de protéines. Suivant la composition de cette couronne, une nanoparticule acquiert une "identité biologique" spécifique qui peut conditionner sa biodistribution ainsi que son éventuelle toxicité.De nombreuses zones d’ombre persistent quant à la connaissance des mécanismes d’adsorption des protéines sur les nanoparticules. Deux caractéristiques physico-chimiques, peu abordées jusqu’à maintenant, ont été étudiées ici : la taille des protéines et la présence de modification post-traductionnelles. Aussi, du fait de leur forte utilisation, nous nous sommes concentrés sur les nanoparticules de silice (SiNPs).L’adsorption d’hémoprotéines, de nature similaire mais de tailles différentes, sur des SiNPs, elles-mêmes de tailles différentes, a été étudiée. Les isothermes d’adsorption et les titrations calorimétriques ont notamment montré qu’il existe une relation entre la taille des protéines et leur affinité pour une surface de silice. Des différences plus fines ont aussi pu être observées selon la taille des nanoparticules. Une analyse structurale des protéines adsorbées a également été effectuée par dichroïsme circulaire et diffusion de neutrons aux petits angles. Les hémoprotéines apparaissent comme des protéines très structurées qui sont peu affectées par l’adsorption. Cependant, bien que la structure quaternaire soit conservée, des modifications structurales sont observables.Des études faites en présence de mélanges de protéines (extraits de protéines de levure) ainsi que de peptides de synthèse ont également montré le rôle important de la diméthylation asymétrique de l’arginine sur l’interaction protéines/SiNPs. L’utilisation d’un panel de techniques expérimentales et de simulations a permis de comprendre le mécanisme responsable de la forte affinité de peptides contenant cette méthylation particulière. De façon plus générale, nos travaux suggèrent que les modifications post-traductionnelles peuvent influencer notablement les interactions de biomolécules avec des surfaces minéralesNanoparticles are ubiquitous in our environment and their presence inside our bodies is now established. Besides, in a biological medium, nanoparticles are spontaneously covered by proteins that form the so-called protein corona. Depending on the corona composition, a nanoparticle will possess a specific "biological identity" conditioning its biodistribution as well as its potential toxicity.Despite being highly studied, many aspects of the protein adsorption mechanisms remain unknown. Here we particularly focused on the influence of two physicochemical characteristics, which had rarely been addressed: protein size and post-translational modifications. Also, because of their intensive use, we worked on silica nanoparticles (SiNPs).We studied the adsorption of hemoproteins on SiNPs, both of them having different sizes. Adsorption isotherms and calorimetry studies showed a relationship between the protein size and its affinity towards silica surfaces. Finer differences could also be observed by varying the SiNPs size. Additionally, structural analyses of adsorbed proteins were performed using circular dichroism and small-angle neutron scattering. The adsorption of hemoproteins, which are well-structured proteins, seems to have little effects on their structure. However, even though the quaternary structure is maintained, structural modifications can be seen.Using yeast protein extracts and synthetic peptides, the major role of arginine asymmetric dimethylation on proteins/SiNPs interaction could be established. The use of experimental and simulation techniques allowed us to understand the mechanism responsible for the high affinity of peptides having this peculiar methylation. As a whole, this work suggests that post-translational modifications can influence considerably the interactions between biomolecules and mineral surfaces
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Chen, Chao. "Self-assembly studies of hybrid nanoparticle-protein cage systems and icosahedral viruses." [Bloomington, Ind.] : Indiana University, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3331353.
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Thesis (Ph.D.)--Indiana University, Dept. of Chemistry, 2008.Title from PDF t.p. (viewed on Jul 27, 2009). Source: Dissertation Abstracts International, Volume: 69-11, Section: B, page: 6818. Adviser: Bogdan Dragnea.
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Capomaccio, Robin. "Interactions nanoparticules-protéines : caractérisation de la couronne protéique." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10323.
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De nos jours, les nanomatériaux sont utilisés dans de très nombreux domaines allant de l’agroalimentaire à la santé en passant par l’industrie automobile et le textile. Dans ce contexte, de nombreuses disciplines comme la métrologie et la nanotoxicologie se sont fortement développées. Dans les milieux biologiques, les protéines interagissent avec les nanoparticules pour former une couronne de protéines. Cette couronne de protéines joue un rôle important dans les interactions des nanoparticules avec leur environnement. Comprendre et caractériser les interactions des nanoparticules avec les protéines permettraient d’améliorer l’utilisation des nanoparticules dans différents domaines, notamment dans celui de la santé. Nous avons d’abord mis au point une méthode de purification des nanoparticules permettant de préserver la structure et la composition de la couronne protéique (Asymmetric flow field-flow fractionation, AF4). Nous avons ensuite caractérisé la couronne protéique associée à ces nanoparticules par plusieurs méthodes telles que la sédimentation centrifuge différentielle, la microscopie électronique, des méthodes basées sur la diffusion de la lumière (diffusion dynamique de la lumière (DLS) et la résonance plasmonique de surface localisée ou non), le dichroïsme circulaire classique et le Synchrotron Radiation Circular Dichroism (SRCD, Diamond, UK). Nous avons montré qu’une couronne d’albumine sérique humaine provoque une augmentation du diamètre hydrodynamique des nanoparticules d’or de 14 à 25,3 nm et une diminution de la densité d’un facteur 2. Ceci nous a permis de calculer que 19 molécules d’albumine en moyenne interagissent avec une nanoparticule. Les spectres de dichroïsme circulaire ont permis d’estimer que l’albumine conserve environ 70% de ses structures hélicoïdales lorsqu’elle est complexée avec les nanoparticules. Nous avons estimé l’affinité avec laquelle les nanoparticules d’or interagissent qui est d’environ 351 nM pour l’albumine et 513 nM pour la transthyrétine qui est riche en brins béta. Nous avons également optimisé une méthode de couplage de l’AF4 à un appareil de mesure de la diffusion dynamique de la lumière (DLS) pour améliorer la précision de la mesure du diamètre hydrodynamique des nanoparticules. Cette méthode précise et flexible permettra de caractériser de nombreuses modifications de surface des nanoparticules comme l’ajout de polyéthylène glycol, utilisées pour la conception de nano-médicamentsNowadays, nanomaterials are used in numerous areas ranging from food to health through cars and textile engineering. In this context, many disciplines such as metrology and nanotoxicology, have been developed. In biological fluids, proteins interact with nanoparticles to form the protein corona, which plays an important role in mediating the interactions of the nanoparticles with their environment. Understanding and characterizing the interactions of nanoparticles with proteins and the corona structure would improve their use in various fields and particularly in the health sector. We have first developed a method based on Asymmetric Flow Field Flow Fractionation (AF4) for purifying gold nanoparticles preserving the structure and composition of the protein corona. Then we have characterized the protein corona associated to these nanoparticles by differential centrifugal sedimentation, electron microscopy, light scattering (dynamic light scattering, DLS), surface plasmon resonance (SPR) and localized SPR and by circular dichroism (classical CD and Synchrotron Radiation Circular Dichroism, SRCD, Diamond, UK). We have shown that human serum albumin corona increased the hydrodynamic diameter of the gold nanoparticles from 14 to 25.3 nm and decreases their density by a factor of 2. This enabled us to calculate that 19 albumin molecules on average interact with a nanoparticle. We have estimated by circular dichroism that albumin maintains about 70% of its helical structures when complexed with nanoparticles. The affinity between gold nanoparticles and proteins, is about 351 nM for albumin and 513 nM for transthyretin, which are enriched in helices and beta strands respectively. We have also optimized a coupling method between the AF4 system and the dynamic light scattering apparatus to improve the measurement accuracy of the hydrodynamic diameter of the nanoparticles. This accurate and flexible method will be helpful to characterize many surface modifications of the nanoparticles such as the addition of polyethylene glycol used for the design of nanodrugs
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Nilsson, Elin. "Proteomics Study of a Designed Nanoparticle-Protein Corona Made of Animal Model Plasma." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167801.
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Nanoparticles are currently finding increasing use as drug delivery systems in the treatment of cancer and other disorders. When nanoparticles are introduced into body fluids, they adsorb proteins forming a coating called protein corona. The protein corona is vital since it controls biological responses of nanoparticles through interactions with cells and biological barriers. Due to the dynamic behaviour of protein-protein and protein-nanoparticle interactions, the protein corona evolves during circulation in the body. This results in difficulties to predict the biological behaviour and outcome of nanoparticles. In this work, it is hypothesised that a nanoparticle-protein corona (NP-PC) enriched in specific proteins could serve as a model to determine if the design and formation of a patient-specific nanodrug-protein corona could offer a novel approach to control nanodrug-protein corona evolution. Through usage of a model nanoparticle and model plasmas and by applying shotgun proteomics and SUrface proteomics, Safety, Targeting, and Uptake (SUSTU), NP-PC proteins were identified and quantified. The results indicate that desirable proteins are maintained in the protein corona surface when nanoparticles with a pre-made corona are introduced into model plasma. This implies that a designed NP-PC would be a strategy to control nanodrug-protein corona evolution, offering a route to improve nanodrug targeting and uptake by cells.
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Agrawal, Amit. "Nanoparticle Probes for Ultrasensitive Biological Detection and Motor Protein Tracking inside Living Cells." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/19798.
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Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)
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Hild, Wolfgang [Verfasser], and Achim [Akademischer Betreuer] Göpferich. "Cellular nanoparticle delivery by G-protein coupled receptors / Wolfgang Hild. Betreuer: Achim Göpferich." Regensburg : Universitätsbibliothek Regensburg, 2010. http://d-nb.info/1023398680/34.
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Lama, Lara. "Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection." Licentiate thesis, KTH, Proteomik och nanobioteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-214065.
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This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins. Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate false positive signals. Paper I shows improved assay sensitivity in a multiplexed vertical flow assay by the application of ultrasonic energy to the gold nanoparticles functionalized with detection antibodies. The ultrasonication of the antibody conjugated gold nanoparticles resulted in a 10 000 fold increase in sensitivity in a 3-plex assay. COMSOL Multiphysics was used to simulate the acoustical energy of the probe used in Paper I for obtaining an indication of the size and direction of the forces acting upon the functionalized gold nanoparticles. In Paper II, it was studied if different gold nanoparticle conjugation methods and colorimetric signal enhancement of the gold nanoparticle conjugates could influence the sensitivity of a paper-based lateral flow microarray assay, targeting cardiac troponin T for the rapid diagnostics of acute myocardial infarction. Ultrasonication and signal enhancement of the detection gold nanoparticles has the potential of improving the sensitivity of paper based assays and expanding their potential future applications.QC 20170911
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Drießen, Marc D. [Verfasser]. "Investigation of nanoparticle toxicity: Characterization of protein corona and evaluation of oxidative stress by protein carbonylation / Marc D. Drießen." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1111558787/34.
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Xie, Mouzhe. "Probing and Modeling Biomolecule-Nanoparticle Interactions by Solution Nuclear Magnetic Resonance Spectroscopy." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1532049249287026.
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Balmori, Pastor Alba. "Harnessing the interaction nanoparticle-protein for the design of smart carriers for drug delivery." Doctoral thesis, Universitat Ramon Llull, 2019. http://hdl.handle.net/10803/667848.
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Les NP han sorgit com prometedors sistemes d'administració de fàrmacs per les seves possibles aplicacions terapèutiques. El procediment comú per a aquest propòsit és dissoldre, encapsular i, finalment, adsorbir o adherir un medicament a la superfície de la NP, evitant el dany no desitjat a les cèl·lules i òrgans sans. De la mateixa manera, les nanopartícules de sílice mesoporoses (MSN) recentment han atret molta atenció en el camp de la nanomedicina per les seves característiques singulars. No obstant això, l'eficàcia terapèutica de les MSN quan s'utilitzen com a sistemes d'administració de fàrmacs sovint es veuen compromeses per l'alliberament previ del fàrmac carregat durant la circulació sanguínia i la manca de capacitat per arribar al destí desitjat. Tenint en compte aquests dos problemes, proposem en aquesta Tesi doctoral la utilització de la "protein corona" per evitar aquest alliberament prematura essent aquesta encaixada al porus. Per aconseguir això, s'han construït HSA-NP carregades amb un fàrmac antitumoral per a la direccionalitat selectiva del fetge utilitzant MSN. L'objectiu principal d'aquest treball és el desenvolupament d'un complex de nanopartícules i proteïnes, capaç de subministrar eficientment un fàrmac terapèutic. Aquest complex ha d'evitar simultàniament l'alliberament prematur i ser capaç de fer targeting al fetge per efecte de la PC.
Per assolir això, en primer lloc, s'ha optimitzat la síntesi de MSN, per tal d'obtenir MSN reproduïbles i amb propietats definides. Després, la caracterització corresponent dels seus trets fisicoquímics va mostrar que, mitjançant el disseny d'un DOE, és possible dilucidar quins paràmetres tenen una major influència en la síntesi de MSN. A més, en el present treball s'ha demostrat que és possible una determinació qualitativa i quantitativa de la "protein corona" mitjançant un ús innovador d'equips analítics; el nanoDSC i l'ITC. Això permet aprofundir en el coneixement i control de PC. També s'ha avaluat l'efecte de la PC en el perfil d'alliberament d'un fàrmac antitumoral. Finalment, s'ha demostrat que la "protein corona" proporciona un alliberament més sostingut del fàrmac a una línia cel·lular de tumor hepàtic, sense afectar la viabilitat cel·lular.
En conclusió, els resultats han demostrat que la relació entre la mida de porus i la mida de la proteïna en els sistemes HSA-MSN determina l'aplicació del sistema. En aquest treball s'ha proposat que, mitjançant l'enginyeria de PC, segons la relació de mida entre el porus i la proteïna es poden fer servir les mateixes proteïnes que formen la "protein corona" per bloquejar la sortida prematura del fàrmac mentre es dirigeix a l'òrgan desitjat.Las NP han surgido como prometedores sistemas de administración de fármacos por sus posibles aplicaciones terapéuticas. El procedimiento común para este propósito es disolver, encapsular y, finalmente, adsorber o adherir un medicamento a la superficie de la NP, evitando el daño no deseado a las células y órganos sanos. De la misma manera, las MSN recientemente han atraído mucha atención en el campo de la nanomedicina debido a sus características singulares. Sin embargo, la eficacia terapéutica de las nanopartículas de sílice mesoporosas (MSN) cuando se utilizan como sistemas de administración de fármacos a menudo se ven comprometidas por la liberación previa del fármaco cargado durante la circulación sanguínea y la falta de capacidad para llegar al destino deseado. Teniendo en cuenta estos dos problemas, proponemos en esta Tesis doctoral la utilización de la “protein corona” para evitar esta liberación prematura siendo ésta encajada en el poro. Para lograr esto, se han construido HSA-NP cargadas con un fármaco antitumoral para la dirección selectiva al hígado utilizando MSN. El objetivo principal de este trabajo es el desarrollo de un complejo de nanopartículas y proteínas, capaz de un suministrar eficientemente un fármaco terapéutico. Este complejo debe evitar simultáneamente la liberación prematura y ser capaz de atacar al hígado por efecto de la PC. Para alcanzar esto, en primer lugar, se ha optimizado la síntesis de MSN, con el fin de obtener MSN reproducibles y con propiedades definidas. Luego, la caracterización correspondiente de sus rasgos fisicoquímicos mostró que, mediante el diseño de un DOE, es posible dilucidar qué parámetros tienen una mayor influencia en la síntesis de MSN. Además, en el presente trabajo se ha demostrado que es posible una determinación cualitativa y cuantitativa de la “protein corona” mediante un uso innovador de equipos analíticos; el nanoDSC y el ITC. Esto permite profundizar en el conocimiento y control de la PC. También se ha evaluado el efecto de la PC en el perfil de liberación de un fármaco antitumoral. Finalmente, se ha demostrado que la “protein corona” proporciona una liberación más sostenida del fármaco a una línea celular de tumor hepático, sin afectar la viabilidad celular. En conclusión, los resultados han demostrado que la relación entre el tamaño de poro y el tamaño de la proteína en los sistemas HSA-MSN determina la aplicación del sistema. En este trabajo se ha propuesto que, mediante la ingeniería de la PC, según la relación de tamaño entre el poro y la proteína se pueden usar las mismas proteínas que forman la “protein corona” para bloquear la salida prematura del fármaco mientras se dirige al órgano deseado.
NPs have gained promise for its potential therapeutic applications as drug delivery systems. The common procedure for this purpose is dissolving, encapsulating, and finally adsorbing or adhering a drug on the NPs surface, avoiding the undesired damage to healthy cells and organs. In the same way, mesoporous silica nanoparticles (MSN) have recently attracted a lot of attention in the nanomedicine field due to their singular characteristics. However, the therapeutic efficiency of MSNs when used as drug delivery systems is often compromised by the pre-release of loaded drug molecules during the blood circulation and the lack of ability to do targeting. Considering these two problems, we propose in this doctoral Thesis the utilization of the widely known protein corona to avoid this premature release by fitting it into the pore. To achieve this, Human Serum Albumin-NP loaded with an antitumor drug for selective liver targeting MSN has been constructed. The main objective of this work is the development of a nanoparticle-protein complex capable of an efficient delivery of a therapeutic drug. This complex should simultaneously avoid premature release and be able to do liver targeting both by effect of the PC. To reach this, first of all the synthesis of MSNs has been optimized, in order to obtain MSNs reproducible and with defined properties. Then, the corresponding characterization of their physicochemical traits showed that through the design of a DOE it is possible to elucidate which parameters have a higher influence on MSN synthesis. Furthermore, it has been here demonstrated that a qualitative and quantitative determination of the protein corona through an innovative use of analytical equipment is possible, the nanoDSC and ITC. This allows the deepening in the PC knowledge and control. It has also been assessed the effect of the protein corona in the release profile of an anti-tumor drug. Finally, the protein corona has been shown to provide a more sustained release of the drug to a liver tumor cell line, without affecting cell viability. In conclusion, results have shown that the relationship between the pore size and the protein size in HSA-MSN systems determine the system’s application. In this work it has been proposed the PC engineering according to the size ratio between the pore and the protein to use the same proteins that form the protein corona as gatekeepers while targeting the desired organ.
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Aubin-Tam, Marie-Eve. "Structure and activity of protein-nanoparticle conjugates: towards a strategy for optimizing the interface." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/54676.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2008.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 130-145).
Nanoparticle-protein conjugates have a variety of applications in imaging, sensing, assembly and control. The nanoparticle-protein interface is made of numerous complex interactions between protein side-chains and the nanoparticle surface, which are likely to affect protein structure and compromise activity. Ribonuclease S and cytochrome c are covalently linked to nanoparticles via attachment to a specific surface cysteine, with the goal of optimizing protein structure and activity, and understanding conditions that minimize non-specific adsorption. Protein behavior is explored as a function of the nanoparticle surface chemistry and material, the density of proteins on the nanoparticle surface, and the position of the labeled site. Ribonuclease S is attached to Au nanoparticles by utilizing its two-piece structure. Enzymatic activity is determined using RNA substrate with a FRET pair. Conjugation lowers the ribonucleatic activity, which is rationalized by the presence of negative charges and steric hindrance which impede RNA in reaching the active site. Cytochrome c is linked to Au and CoFe204 nanoparticles. The protein is denatured when the nanoparticle ligands are charged, but remains folded when neutral. The presence of salt in the buffer improves folding. This indicates that electrostatic interactions of charged amino acids with the charged ligands are prone to lead to protein denaturation. The attachment site can be controlled by mutations of surface residues to cysteines. Protein unfolding is more severe for nanoparticle attached in the vicinity of charged amino acids. Molecular dynamics simulations of the conjugate reveal that electrostatic interactions with· the nanoparticle ligand lead to local unfolding of [alpha]-helices of cyt c. Furthermore, the nanoparticle induces more structural disturbance when it is attached on the N- and C-terminal [alpha]-helices foldon, which is the most stable motif of cyt c and the most essential for folding.
by Marie-Eve Aubin-Tam.
Ph.D.
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Bruzas, Ian R. "Biocompatible noble metal nanoparticle substrates for bioanalytical and biophysical analysis of protein and lipids." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1553250462519941.
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Hassan, Anwar I. "Development of ¹⁹F NMR Methods for the Study of GlpG Rhomboid Protease in Detergents and Lipid Nanoparticle Systems." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42521.
Full textAbstract:
Rhomboids are a family of intramembrane serine proteases that cleave transmembrane protein substrates within the lipid membrane. They are involved in a wide range of biological processes, including signal transduction, parasite invasion, bacterial quorum sensing and apoptosis. While previous X-ray crystal structures and functional studies have provided some detailed insights into the mechanism of intramembrane hydrolysis, it is still not clear how the transmembrane substrate can gain access into the active site from the lipid environment. While several modes of action have been suggested, one hypothesis proposes a lateral movement of the fifth transmembrane helix, causing a displacement that would allow transmembrane substrates to enter the rhomboid active site. A powerful method that has the potential to yield insights into rhomboid dynamics is solution NMR; however, the large size of rhomboid protease samples has complicated conventional methods typically used to assess protein structure and dynamics. ¹⁹F NMR could allow the study of rhomboid conformational dynamics by providing a simplified spectrum with high sensitivity to changes in local chemical environments. In this thesis various methods of ¹⁹F incorporation were evaluated for utility in studying rhomboid conformational dynamics, focusing on the GlpG rhomboid from E. coli. First, GlpG samples were prepared with ¹⁹F incorporated into tryptophan sidechains, and 1D ¹⁹F NMR spectra were acquired. While spectra with decent spectral dispersion were obtained, the assignment process was complicated by low signal-to-noise, and multiple changes in the spectrum introduced by the mutation. Chemoselective labelling of cysteine residues with probes containing a trifluoromethyl group was also investigated and found to give rise to well resolved ¹⁹F NMR spectra with promising characteristics. In addition, protocols for incorporation of trifluoromethyl-phenylalanine using unnatural amino acid incorporation at introduced amber codon sites were also explored, since one of the long-term goals of this work is to study ¹⁹F-labelled GlpG in its native lipid environment. For this purpose, some protocol development was also performed to introduce GlpG into lipid nanoparticles using styrene maleic acid co-block polymers. However, low expression yields of trifluoromethyl-phenylalanine-labelled GlpG and the large size of the lipid nanoparticles are not yet compatible with solution NMR. Nonetheless, this thesis lays the groundwork for further development of these samples to allow the future study of conformational exchange of GlpG in native lipid membranes.
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Shah, Bhavik P. "Targeting Fat-Sensitive Pathways In Enteroendocrine Cells Using Nanoparticle-Mediated Drug Delivery." DigitalCommons@USU, 2009. https://digitalcommons.usu.edu/etd/432.
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The current epidemic of obesity has been linked to an increase in fat intake associated with the Western diet. Nutrient-induced stimulation of enteroendocrine cells in the small intestine leads to the release of hormones that contribute to satiety and the control of food intake. In particular, ingested fat, specifically in the form of free fatty acids, is potent activator of enteroendocrine cells in the proximal small intestine. However, the underlying signaling cascade that free fatty acids initiate in these enteroendocrine cells, which leads to secretion of satiety hormones, is not known. In general, my research is focused on identifying nutrient-responsive pathways in enteroendocrine cells involved with the release of satiety signals and using this information to begin to develop novel drug delivery strategies to reduce food intake. In general, my results revealed that activation of the fatty acid receptor GPR120 was ecessary for the linoleic acid-induced intracellular calcium rise, a necessary precursor for hormone release. Using patch clamp recording, I discovered that linoleic acid activated enteroendocrine cells by inducing membrane depolarization, a process requiring the calcium-activated, monovalent cation permeable channel TRPM5, which is activated downstream of GPR120. To validate the unexpected finding that TRPM5 was involved in fattyacid signaling, I performed experiments using bitter compounds, whose transduction pathway is known to involve TRPM5. Enteroendocrine cells express the bitter taste receptors and release cholecystokinin in response to bitter stimuli, suggesting the probable role of gut in initiation of protective behavior against ingestion of potentially harmful substances. Armed with the data on the specifics of the fatty acid transduction, I performed experiments using nanoparticles to determine their utility for delivering pharmaceuticals specifically to the enteroendocrine cells. I fabricated and characterized PLGA nanoparticles and performed intracellular uptake studies in order to optimally delivery payloads inside cells. Finally, I validated their use by using cell-based assays to determine the effects of internalized PLGA nanoparticles on ion channels and signaling pathways involved in CCK release. Taken together, this dissertation research has identified the signaling pathways (pharmacological targets) involved in fatty acid-mediated satiety hormone release and validated the potential therapeutic use of nanoparticle-mediated drug delivery for the eventual control of food intake.
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Ouyang, Wuye. "Dispersed and deposited polyelectrolyte complexes and their interactions to chiral compounds and proteins." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1233841492089-61523.
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Polyelectrolyte complexation is a rapidly growing field with applications in functional multilayer (PEM) and nanoparticle (PEC) generation, where PEM films are deposited using Layer-by-Layer technique initiated by Decher and PECs are prepared using mixing-centrifugation technique initiated by our group. Its advantages (e.g. easy preparation) result in various applications in aqueous solution, especially in pharmaceutical and biomedical fields. Therefore, the objectives in this study are to explore interesting applications of polyelectrolyte complexation in the field of low molecular chiral compound and high molecular protein binding. Due to the rapidly growing demands for preparing optically pure compounds in the pharmaceutical field, herein, enantiospecific PEM and PEC were prepared using chiral polyelectrolytes (e.g. homo-polypeptide) and their ability of chiral recognition was investigated by ATR-FTIR, UV/Vis etc.. Chiral PEM and PEC showed pronounced enantiospecificity for both small (amino acids, vitamin) and large (protein) chiral compounds. This chiral recognition is performed by a diffusion process of chiral compounds into PEM based on the structures of chiral selector (PEM, PEC) and chiral probes (chiral compounds). However, the influences, e.g. pH value, ionic strength, surface orientation etc., were found to affect significantly the enantiospecificity. Beside planar substrates, porous membranes (e.g. PTFE) were modified using chiral PEM and successfully applied in enantiospecific permeation. Additionally, protein binding properties of PEC particle dispersions or PEC particle films were also studied. Due to the properties of polyelectrolytes used for PEC (e.g. molecular weight, charge density) and proteins (e.g. isoelectric point, size, hydrophobicity), PEC showed different uptake characteristics towards different proteins. Electrostatic and hydrophobic interaction as well as counterion release force were considered as possible driving forces for protein binding.
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Fleischer, Candace C. "A molecular snapshot of charged nanoparticles in the cellular environment." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53632.
Full textAbstract:
Nanoparticles are promising platforms for biomedical applications ranging from diagnostic tools to therapeutic delivery agents. During the course of these applications, nanoparticles are exposed to a complex mixture of extracellular serum proteins that nonspecifically adsorb onto the surface. The resulting protein layer, or protein "corona," creates an interface between nanoparticles and the biological environment. Protecting the nanoparticle surface can reduce protein adsorption, but complete inhibition remains a challenge. As a result, the corona, rather than the nanoparticle itself, mediates the cellular response to the nanoparticle. The following dissertation describes the fundamental characterization of the cellular binding of charged nanoparticles, interactions of protein-nanoparticle complexes with cellular receptors, and the structural and thermodynamic properties of adsorbed corona proteins.
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Soleilhac, Antonin. "Thermographie multi-échelle par méthodes optiques sur gouttelettes et bulles micrométriques : applications aux nanoparticules irradiées par laser ultra-intense et aux édifices biomoléculaires en cours de déshydratation." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1192/document.
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Ce travail de thèse a eu pour but d'étudier la thermodynamique de petits objets (nanoparticules ou édifices biomoléculaires) dans des milieux fortement hors-équilibre. Deux milieux miroirs ont été explorés, à savoir des gouttelettes micrométriques chargées et des microbulles générées sous irradiation laser intense. Ces deux systèmes sont par nature difficiles à sonder par des méthodes traditionnelles, c'est pourquoi, une thermographie innovante multi-échelle par méthodes optiques a été développée. Ainsi, une thermographie locale (au sein des nano-objets) et globale (dans le milieu environnant) a été réalisée. Cette thermographie utilise la fluorescence induite par laser - méthode simple, non invasive et efficace pour fournir une mesure de température avec une bonne résolution spatiale, temporelle et thermique - grâce à des colorants thermochromiques directement en solution dans le milieu ou bien incorporés dans les nano-objets. Cette thermographie est également complétée avec d'autres mesures physiques, notamment la taille des gouttelettes et des bulles micrométriques pour aller vers une étude thermodynamique exhaustive de ces systèmes. Ces études thermodynamiques ont été menées autour des deux thématiques : Edifices biomoléculaires dans des gouttelettes micrométriques chargées en cours de déshydratation. Les sources électrospray, devenues un outil incontournable en spectrométrie de masse, présentent une thermodynamique riche et encore mal comprise. Des mesures de taille et de température de gouttelettes micrométriques chargées en cours d'évaporation ont pu être confrontées dans le but d'avoir une description thermodynamique complète d'une source électrospray. De plus, afin de pouvoir suivre la conformation des édifices biomoléculaires (protéines) en cours de déshydratation, le concept d'anisotropie de fluorescence comme sonde conformationnelle a été validé en solution et pourra être transposé pour une analyse in situ dans la plume de l'électrospray.Nanoparticules dans des microbulles générées sous irradiation laser intense. Il s'agit ici d'étudier l'influence de nanoparticules lors de la génération de microbulles par nano-cavitation induite par irradiation laser intense, thématique au coeur du projet « ERTIGO ». Le principal objectif scientifique de ce projet a été d'obtenir une compréhension des mécanismes d'absorption d'une solution contenant des nanoparticules irradiées. A cet effet, une source lumineuse atypique (laser aléatoire) a été employéeThe aim of this thesis was to study the thermodynamics of small objects (nanoparticles or biomolecule) in out-of-equilibrium media. Two mirror media were explored, namely charged micrometric droplets and microbubbles generated under intense laser irradiation. By nature, these two systems are difficult to probe by traditional methods, which is why an innovative multi-scale thermography by optical methods has been developed. Thus, a local (within the nano-objects) and a global (in the surrounding medium) thermography were carried out. Such thermography uses Laser-Induced Fluorescence - a simple, non-invasive and efficient method for providing temperature measurements with good spatial, temporal and thermal resolution - using thermochromic dyes directly in solution or incorporated into nano- objects. This thermography is also supplemented with other physical measurements, in particular the size of microdroplets and microbubbles, towards a complete thermodynamic study of these systems. These thermodynamic studies were carried out around the two following themes: Biomolecular structures in charged microdroplets during dehydration. Electrospray sources, which have become an essential tool in mass spectrometry, present a rich and still poorly understood thermodynamics. Measurements of the size and temperature of charged micrometric droplets during evaporation have been possible in order to obtain a complete thermodynamic description of an electrospray source. Moreover, in order to be able to follow the conformation of biomolecules (proteins) during dehydration, the concept of fluorescence anisotropy as a conformational probe has been validated in solution and can be transposed for an in situ analysis into the electrospray plume. Nanoparticles in microbubbles generated by intense laser irradiation. Here, we want to study the influence of nanoparticles during the generation of microbubbles by nano-cavitation induced by intense laser irradiation. This thematic is at the heart of the project "ERTIGO". The main scientific objective of this project was to obtain an understanding of the absorption mechanisms of a solution containing irradiated nanoparticles. For this purpose, an atypical light source (random laser) has been used in order to be able to illustrate this complex out-of-equilibrium system as a function of time by optical microscopy. In parallel, a local measurement of the temperature of the nanoparticles is envisaged
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Vannoy, Charles Harvey. "Behavioral Effects of Functionalized CdSe/ZnS Quantum Dots in Self-Organization and Protein Fibrillation." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/431.
Full textAbstract:
Advances in recent nanoscience technologies have generated a new compilation of biocompatible, fluorescent nanoparticles derived from semiconductor quantum dots (QDs). QDs are extremely small in size and possess very large surface areas, which gives them unique physical properties and applications distinct from those of bulk systems. When exposed to biological fluid, these QDs may become coated with proteins and other biomolecules given their dynamic nature. These protein-QD systems may affect or enhance the changes in protein structure and stability, leading to the destruction of biological function. It is believed that these QDs can act as nucleation centers and subsequently promote protein fibril formation. Protein fibrillation is closely associated with many fatal human diseases, including neurodegenerative diseases and a variety of systemic amyloidoses. This topic of protein-QD interaction brings about many key issues and concerns, especially with respect to the potential risks to human health and the environment. Herein, the behavioral effects of dihydrolipoic acid (DHLA)-capped CdSe/ZnS (core/shell) QDs in hen egg-white lysozyme (HEWL) and human serum albumin (HSA) protein systems were systematically analyzed. This study gives rise to a better understanding of the potentially useful application of these protein-QD systems in nanobiotechnology and nanomedicine as a bioimaging tool and/or as a reference for controlled biological self-assembly processes.
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Morgan, Sherif, Raymond Nagle, and Lee Cranmer. "Serum protein acidic and rich in cysteine (SPARC) as a prognostic marker in soft tissue sarcomas." BioMed Central, 2014. http://hdl.handle.net/10150/610120.
Full textAbstract:
BACKGROUND:Serum protein acidic and rich in cysteine (SPARC) is a matricellular secreted glycoprotein that performs several cellular functions and has been implicated in tumorigenesis in a variety of tumor types. The chemotherapeutic agent nanoparticle albumin-encapsulated (NAB)-paclitaxel has been postulated to exploit SPARC expression to target neoplastic cells. SPARC's role, and potentially the role of NAB-paclitaxel, in the highly heterogeneous class of soft-tissue sarcomas (STS) has not been investigated. Our objective was to explore the pattern of SPARC expression and its prognostic significance in STS.METHODS:27 tissue specimens representing various STS histologies were stained for SPARC expression by immunohistochemistry (IHC). Staining intensity was scored blindly. Survival was determined from patients' medical records and analyzed using Kaplan-Meier and log-rank with respect to SPARC expression level.RESULTS:Elevated SPARC expression was observed in 15/27 (56%) specimens. Overall patient survival segregated strongly based on levels of SPARC expression. Patients who expressed low-to-moderate levels of SPARC exhibited median survival of 22.1months, while the median survival of patients with moderate-to-high expression levels was 4.4months (log rankp=0.0016).CONCLUSIONS:SPARC expression is elevated in a significant proportion of STS specimens analyzed in this study, but it does not appear to correlate with specific STS histologies. Given our limited sample size, we cannot draw definitive conclusions regarding association of SPARC with STS subtype. Overall survival segregates strongly by degree of SPARC expression, with elevated expression being adverse. If validated in a larger study, our results suggest that trials in STS with agents potentially targeting SPARC, such as NAB-paclitaxel, should be stratified by SPARC expression level.
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Xu, Zhikun. "Design of protein nanoparticles for cell targeting and blood brain barrier crossing." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/308333.
Full textAbstract:
En estos últimos años, muchos nuevos materiales han sido involucrados en los campos de las nanotecnologías, y especialmente en nanomedicina. Las nanoparticulas formadas por proteínas son un ejemplo de bio-material que está atrayendo cada vez más la atención debido a sus características tanto biológicas como estructurales, como por ekjemplo su baja toxicidad, baja inmunogenicidad y su biodegradabilidad. Además, a través de la ingeniería genética, estas proteínas se pueden diseñar para que formen multímeros estructuras con la capacidad de auto-ensamblarse en una estructura similar a las capsides virales. Estas propiedades permiten el utilizo de estas nanoparticulas proteicas en el diseño de vectores para la entrega de fármacos o genes terapéuticos, el diseño de vacunas y kits de diagnósticos. La entrega de moléculas terapéuticas supones la superación de barreras tanto extracelulares como intracelulares. Gracias a la exposición de ligandos en la superficie externa y a su tamaño reducido, las nanopartículas pueden superar estas barreras como, por ejemplo, la Barrera Hematoencefalica (BBB).
En este contexto, hemos desarrollado unas nanopartículas proteicas auto-ensamblables con especificidad para los receptores LDLR, con el propósito de utilizarlas como vehículos para el tratamiento de enfermedades en el Sistema Nervioso Central (SNC). Cuatro diferentes ligandos específicos para los LDLR se fusionaron a la proteína GFP -His; entre ellos, solamente el ligando ApoB fue capaz de promover la formación de nanopartículas proteicas por interacciones intermoleculares entre el mismo ApoB, la cola de histidinas y los monómeros vecinos. Estas nanoparticula han demostrado una buena internalización en líneas celulares LRDR+, en experimentos in vitro. Sin embargo, cuando se probó en un modelo in vivo, solamente dos proteínas ligandos que no forman nano-particulas se han detectado en puntos de tiempo post-administración cortos. Esto indicaría que la formación de nano-estructuras no favorece la acumulación en el SNC.
Paralelamente, se estudiaron nano-partículas proteicas dirigidas hacia el receptor CXCR4, un receptor de superficie celular expuesto en la superficie celular de las células metástaticas de cáncer colorrectal. En este estudio la proteína T22-IRFP-HIS fue elegida para sustituir a la ApoB-GFP-HIS y también se observó la formación de nanoparticulas autoensamblables que además poseen una alta internalización en las células CXCR4+. Esto indica que la proteína andamio no afecta a la formación de nanopartículas, ni afecta a la especificidad del ligando de direccionamiento. La fuerza que guía a la formación de nanopartículas se basa principalmente en las interacciones electrostáticas entre monómeros de proteínas. Además este fenómeno puede ser interrumpido y revertido por la presencia de alta concentración de sal. De hecho, cuando las nanoparticulas de T22-IRFP-HIS se dializan a un tampón con alta concentración de sal, las estructuras se desensamblan en monómeros y además reducen su eficiencia de penetrabilidad celular. Esto demostraría que el tamaño y tal vez la multivalencia de la nanopartículas proteicas frente a la monovalencia de los monómeros es un factor clave para el reconocimiento celular especifico y la internalización de las proteínas.With the development of nanotechnology and nanomedicine, more and more materials have been involved in these fields, protein based nanoparticles have become another biomaterial that is developing fast and attracts growing attention. They are non-toxic, low-antigenic, biodegradable, metabolizable, and with genetic engineering, it is easy to modify their structure, surface charge, to allow heterologous ligand display, to improve stability and more importantly, proteins can be designed to form multimeric structures with the ability to self-assemble in a similar way as viral capsid proteins do. These properties enable protein particles to be widely used in targeting and delivery of therapeutic drugs, vaccine designing, diagnosis, and gene therapy.Nanoparticle-mediated targeting delivery is also widely used when overcoming barriers in human body, especially in the BBB. Nanoparticles could help drugs to cross the BBB because they have suitable size and could exhibit targeting ligands on the surface. These ligands could interact with receptors at the BBB and then transport nanoparticles across BBB by receptor mediated transcytosis. Based on that, the first part of this context is aim to construct self-assembled protein nanoparticles targeting on LDLR (which is a high affinity binding site in brain capillaries), with the purpose of using nanostructured materials as vehicles for the systemic treatment of CNS diseases. Four different LDLR specific ligands were fused to GFP protein and His tag; among those, only ApoB ligand, was able to promote the formation of protein nanoparticles by intermolecular interactions involving the ApoB ligand and the His tag of a neighboring monomer. This ApoB empowered protein nanoparticle showed higher internalization ability on LDLR+ cells, and higher permeability in BBB in vitro model. However, when tested those proteins displaying LDLR ligands in an in vivo model, two proteins which were not able to form nanoparticle accumulated in at short post-administration time points, indicating that the nanoparticulate form is not favoring the accumulation apart from preventing the transient accumulation. This work brings up new concepts of BBB crossing properties by using functional protein nanoparticles. The second part of this context is aimed to produce size-controllable protein nanoparticles towards CXCR4 receptor (CXCR4, a cell surface receptor marker associated with metastasis-forming colorectal cancer cells and other human pathologies) expressing cells. In this part, a new scaffold protein iRFP was chosen to replace GFP, we determined that it could also self-assemble into nanoparticles, showing high penetration into CXCR4+ cells. This indicates that the o scaffold protein has neither affect on the formation of nanoparticles, nor on the ligand targeting ability. The force which drives nanoparticle formation mainly is based on electrostatic interactions between protein monomers, and this can be interrupted by the presence of high salt concentration. Moreover, when this T22 empowered nanoparticle is transferred to a high salt concentration buffer, protein naaparticles disassemble into monomers reducing its cell penetrability efficiency, proving again that size and perhaps the multivalency of the protein nanoparticle versus the monovalency of protein monomers is a key factor in receptor mediated cell targeting and penetration.
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Boström, Fredrik. "Single-cycle kinetics for QCM biosensors for high throughput nanoparticle characterization application." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-298942.
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Characterizing nanoparticles to be able to understand how they functions in the body is important for development of drugs. Furthermore with increasing number of nanoparticle product the nanotoxicity of nanoparticles is important to understand. This report is a part of the EU-project Nanoclassifier which purpose is to “develop a cost effective, high throughput screening platform for characterization of the bionanointerface and its cell-binding partners”. Single-cycle kinetic was used to determine the number of binding epitopes on polystyrene nanoparticle with transferrin corona. The number of available epitopes describes how active the Nanoparticle will be in the body. For this purpose Single-cycle kinetic methodology was successfully used on nanoparticles. Single-cycle kinetic methodology has great potential to become the standard method for high throughput nanoparticle epitope characterization.
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Ouyang, Wuye. "Dispersed and deposited polyelectrolyte complexes and their interactions to chiral compounds and proteins." Doctoral thesis, Technische Universität Dresden, 2008. https://tud.qucosa.de/id/qucosa%3A23825.
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Polyelectrolyte complexation is a rapidly growing field with applications in functional multilayer (PEM) and nanoparticle (PEC) generation, where PEM films are deposited using Layer-by-Layer technique initiated by Decher and PECs are prepared using mixing-centrifugation technique initiated by our group. Its advantages (e.g. easy preparation) result in various applications in aqueous solution, especially in pharmaceutical and biomedical fields. Therefore, the objectives in this study are to explore interesting applications of polyelectrolyte complexation in the field of low molecular chiral compound and high molecular protein binding. Due to the rapidly growing demands for preparing optically pure compounds in the pharmaceutical field, herein, enantiospecific PEM and PEC were prepared using chiral polyelectrolytes (e.g. homo-polypeptide) and their ability of chiral recognition was investigated by ATR-FTIR, UV/Vis etc.. Chiral PEM and PEC showed pronounced enantiospecificity for both small (amino acids, vitamin) and large (protein) chiral compounds. This chiral recognition is performed by a diffusion process of chiral compounds into PEM based on the structures of chiral selector (PEM, PEC) and chiral probes (chiral compounds). However, the influences, e.g. pH value, ionic strength, surface orientation etc., were found to affect significantly the enantiospecificity. Beside planar substrates, porous membranes (e.g. PTFE) were modified using chiral PEM and successfully applied in enantiospecific permeation. Additionally, protein binding properties of PEC particle dispersions or PEC particle films were also studied. Due to the properties of polyelectrolytes used for PEC (e.g. molecular weight, charge density) and proteins (e.g. isoelectric point, size, hydrophobicity), PEC showed different uptake characteristics towards different proteins. Electrostatic and hydrophobic interaction as well as counterion release force were considered as possible driving forces for protein binding.
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Yang, Fan [Verfasser], and Frank [Akademischer Betreuer] Rösl. "Re-engineering a Nanoparticle Human Papillomavirus Prophylactic Vaccine Antigen Based on the Minor Capsid Protein L2 / Fan Yang ; Betreuer: Frank Rösl." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/121816798X/34.
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Díaz, Ocaña Raquel. "Recombinant self-assembling nanoparticles for cancer therapy based on toxin and venom compounds." Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670483.
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La plataforma desenvolupada d’enginyeria de proteïnes auto-acoblables permet dissenyar nanopartícules únicament proteiques (NPs) capaces d’atacar i actuar selectivament sobre les cèl.lules canceroses mitjançant la interacció amb receptors que es sobreexpressen. Les estructures esfèriques estables de les NPs desenvolupades i la seva mida adequada, en combinació amb els pèptids d’orientació , milloren la seva especificitat. A més, la incorporació de segments de toxina i verí ha millorat els efectes terapèutics d’aquestes estructures que són totalment biocompatibles i que no tenen cap portador extern o material agregat, complint d’aquesta manera amb el nou concepte per a medicaments de precisió, que involucra un fàrmac recombinant lliure de vehicle, auto-acoblat, auto-dirigit i eficient.
Una versió modificada de la cadena catalítica ricina A, amb la capacitat de disminuir els efectes secundaris no desitjats de la síndrome de vessament vascular però conservant la seva citotoxicitat natural, es va adaptar a la plataforma de proteïnes. El disseny es va desenvolupar amb el pèptid T22, que s’uneix a CXCR4, en l’extrem N-terminal, i una cua d’histidines a la terminal-C, en combinació amb un fragment del lloc escindible de furina per alliberar la proteïna intracel.lularment, i una seqüència KDEL per evitar la secreció del reticle endoplàsmic. Les NPs de cadena de ricina A solubles purificades dirigides a CXCR4 amb un diàmetre mitjà de 11 nm, van assolir un increment de 100 vegades en la seva citotoxicitat amb un IC50 de 13 ± 0,5 x 10 -9 M en cèl.lules HeLa. També es van produir per mètodes recombinants i es van purificar cossos d’inclusió insolubles de 400-600 nm, amb resultats citotòxics parcials. El mecanisme d’entrada dependent del receptor d’T22-MRTA-H6 es va verificar i avaluar en un model de ratolí amb leucèmia mieloide aguda (AML) mitjançant la injecció sistèmica a la vena de la cua on es va verificar un bloqueig important de les cèl.lules leucèmiques sense toxicitat sistèmica o histològica lateral en els òrgans sans.
De manera similar, la clorotoxina (CTX) també es va incorporar a la plataforma de proteïnes per tal d’aprofitar la seva d’orientació i efecte terapèutic en glioblastoma (GBM), ambdues funcions en un sol pèptid. Es van dissenyar dues versions que s’uneixen a la proteïna anexina-2 i la metaloproteinasa de matriu MMP-2; CTX-GFP-H6 i CTX-KRKRK-GFP-H6. Les NPs solubles d’un diàmetre mitjà de 12 nm es van incubar en cèl·lules HeLa, sobreexpressant annexina-2, i en cèl.lules U87MG, sobreexpressant MMP2. Les dues versions eren completament fluorescents, però CTX-GFP-H6 va presentar efectes citotòxics lleus, mentre que CTX-KRKRK-GFP-H6 va mostrar ser més citotòxic en les cèl.lules U87MG que en les cèl.lules HeLa. L’afinitat selectiva de CTX es va confirmar mitjançant l’avaluació de la seva selectivitat utilitzant anticossos monoclonals i un sèrum policlonal contra la proteïna de la superfície cel.lular, actuant com un receptor de la CTX.La plataforma desarrollada de ingeniería de proteínas autoensamblables permite diseñar nanopartículas únicamente proteicas (NPs) capaces de atacar y actuar selectivamente sobre las células cancerosas mediante la interacción con receptores que se sobreexpresan. Las estructuras esféricas estables de las NPs desarrolladas y su tamaño adecuado, en combinación con los péptidos de direccionamiento involucrados, mejoran su especificidad. Además, la novedosa incorporación de segmentos de toxina y veneno ha mejorado los efectos terapéuticos de estas estructuras que son totalmente biocompatibles y que no tienen ningún portador externo o material agregado, cumpliendo de esta manera con el concepto emergente para medicamentos de precisión que involucra un fármaco recombinante libre de vehículo, autoensamblado, auto-dirigido y eficiente. Una versión modificada de la cadena catalítica de ricina A, con la capacidad de disminuir los efectos secundarios no deseados del síndrome de derrame vascular, pero conservando su citotoxicidad natural, se adaptó a la plataforma de proteínas. El diseño se desarrolló con el péptido T22, que se une a CXCR4, en el extremo N-terminal, y una cola de histidinas en el extremo C-terminal, en combinación con un fragmento del sitio escindible de furina para liberar la proteína intracelularmente, y una secuencia KDEL para evitar secreción del retículo endoplásmico. Las NPs de cadena de ricina A solubles purificadas dirigidas a CXCR4, con un diámetro promedio de 11 nm, alcanzaron un incremento de 100 veces en su citotoxicidad con un IC50 de 13 ± 0,5 x 10 -9 M en células HeLa. Pero también se produjeron por métodos recombinantes y se purificaron cuerpos de inclusión insolubles de 400-600 nm, con resultados citotóxicos parciales. El mecanismo de entrada dependiente del receptor de T22-mRTA-H6 se verificó y evaluó en un modelo de ratón con leucemia mieloide aguda (AML) mediante la inyección sistémica en la vena de la cola, donde se verificó un bloqueo importante de las células leucémicas sin toxicidad sistémica o histológica lateral en los órganos sanos. De manera similar, la clorotoxina (CTX) también se incorporó a la plataforma de proteínas con el fin de aprovechar su direccionamiento y efecto terapéutico en glioblastoma (GBM), ambas funciones en un solo péptido. Se diseñaron dos versiones que se unen a la proteína anexina-2 y la metaloproteinasa de matriz MMP-2; CTX-GFP-H6 y CTX-KRKRK-GFP-H6. Lss NPs solubles, de un diámetro promedio de 12 nm, se incubaron en células HeLa sobreexpresando anexina-2, y en células U87MG, sobreexpresando MMP2. Ambas versiones eran completamente fluorescentes, pero CTX-GFP-H6 presentó efectos citotóxicos leves, mientras que CTX-KRKRK-GFP-H6 mostró ser más citotóxico en las células U87MG que en las células HeLa. La afinidad selectiva de CTX se confirmó mediante la evaluación de su direccionamiento utilizando anticuerpos monoclonales y un suero policlonal contra la proteína de la superficie celular, actuando como un receptor de la CTX.
The developed self-assembling platform allows the engineering of protein-only nanoparticles (NPs) capable to target and act selectively over cancer cells by means of the interaction with overexpressed receptors. The stability of the spherical NP structures and their adequate size, in combination with the involved targeting peptides, enhance their specificity. Also, the novel incorporation of toxin and venom segments have improved the therapeutic effects of these fully biocompatible materials, without the need of any external carrier or added material, thus fulfilling the newfangled concept for precision medicines that involve self-assembled, self-targeted and efficient vehicle-free recombinant drugs. A modified version of the catalytic ricin A chain, with the ability to diminish the undesired vascular leak syndrome side effects but retaining its natural cytotoxicity, was adapted to the protein platform. The design was developed with the peptide T22 in the N-terminal, which binds CXCR4, and a his-tag in the C-terminal. This was combined with a furin cleavable site fragment in order to release the protein intracellularly, and a KDEL sequence to avoid endoplasmic reticulum secretion. Purified soluble CXCR4-targeted ricin A chain NPs with an average diameter of 11 nm, reached a 100-fold cytotoxic improvement with an IC50 of 13 ± 0.5 x 10 -9 M in HeLa cells. Also, insoluble 400-600 nm inclusion bodies were produced by recombinant methods and purified, with partial cytotoxic results. The receptor-dependent mechanism of T22-mRTA-H6 was verified and evaluated in an acute myeloid leukemia (AML) mouse model by systemic administration through a vein tail injection where an important blockage of the leukemic cells was verified without side systemic or histological toxicity in healthy organs. In a similar way, chlorotoxin (CTX) was also incorporated to the protein platform in order to take advantage of its targeting and therapeutic effect in glioblastoma (GBM), both functions in one peptide. Two versions that target protein Annexin-2 and the matrix metalloproteinase MMP-2 were engineered, namely CTX-GFP-H6 and CTX-KRKRK-GFP-H6. The soluble NPs of an average dimeter of 12 nm were incubated with HeLa cells, overexpressing annexin-2, and in U87MG cells, overexpressing MMP2. Both versions were fully fluorescent but CTX-GFP-H6 presented mild cytotoxic effects, whereas CTX-KRKRK-GFP-H6 showed to be more cytotoxic in U87MG cells than in HeLa cells. The selective affinity of CTX was confirmed by means of evaluating its targeting using a monoclonal antibody and a polyclonal serum against the cell surface protein, acting as a CTX receptor.
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Schneider, René. "A novel parabolic prism-type TIR microscope to study gold nanoparticle-loaded kinesin-1 motors with nanometer precision." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-110212.
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Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions.
The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision.
Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum.
Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future.
To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate.
To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks.
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Zhang, Fan. "The Mussel Adhesive Protein (Mefp-1) : A GREEN Corrosion Inhibitor." Doctoral thesis, KTH, Yt- och korrosionsvetenskap, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-123489.
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Corrosion of metallic materials is a natural process, and our study shows that even in an alkaline environment severe corrosion may occur on a carbon steel surface. While corrosion cannot be stopped it can be retarded. Many of the traditional anti-corrosion approaches such as the chromate process are effective but hazardous to the environment and human health. Mefp-1, a protein derived from blue mussel byssus, is well known for its extraordinary adhesion and film forming properties. Moreover, it has been reported that Mefp-1 confers a certain corrosion protection for stainless steel. All these facts indicate that this protein may be developed into corrosion inhibitors with ‘green’, ‘effective’ and ‘smart’ properties. In this study, a range of surface-sensitive techniques have been used to investigate adsorption kinetics, film forming and film compaction mechanisms of Mefp-1. In situ atomic force microscopy (AFM) enables the protein adsorption on substrates to be visualized, whereas the ex situ AFM facilitates the characterization of micro- and nano-structures of the protein films. In situ Peak Force AFM can be used to determine nano-mechanical properties of the surface layers. The quartz crystal microbalance with dissipation monitoring (QCM-D) was used to reveal the build-up of the Mefp-1 film on substrates and measure the viscoelastic properties of the adsorbed film. Analytical techniques and theoretical calculations were applied to gain insights into the formation and compaction processes such as oxidation and complexation of pre-formed Mefp-1 films. The electron probe micro analyzer (EPMA) and X-ray photoelectron spectroscopy (XPS) were utilized to obtain the chemical composition of the surface layer. Electrochemical impedance spectroscopy (EIS) measurements were performed to evaluate the corrosion inhibition efficiency of different forms of Mefp-1 on carbon steel substrates. The results demonstrate that Mefp-1 adsorbs on carbon steel surfaces across a broad pH interval, and it forms a continuous film covering the substrate providing a certain extent of corrosion protection. At a higher pH, the adsorption is faster and the formed film is more compact. At neutral pH, results on the iron substrate suggest an initially fast adsorption, with the molecules oriented preferentially parallel to the surface, followed by a structural change within the film leading to molecules extending towards solution. Both oxidation and complexation of the Mefp-1 can lead to the compaction of the protein films. Addition of Fe3+ induces a transition from an extended and soft protein layer to a denser and stiffer one by enhancing the formation of tri-Fe3+/catechol complexes in the surface film, leading to water removal and film compaction. Exposure to a NaIO4 solution results in the cross-linking of Mefp-1, which also results in a significant compaction of the pre-formed protein film. Mefp-1 is an effective corrosion inhibitor for carbon steel when added to an acidic solution, and the inhibition efficiency increases with time. As a film-forming corrosion inhibitor, the pre-formed Mefp-1 film provides a certain level of corrosion protection for short term applications, and the protection efficiency can be significantly enhanced by the film compaction processes. For the long term applications, a thin film composed of Mefp-1 and ceria nanoparticles was developed. The deposited Mefp-1/ceria composite film contains micro-sized aggregates of Mefp-1/Fe3+ complexes and CeO2 particles. The Mefp-1/ceria film may promote the further oxidation of ferrous oxides, and the corrosion resistance increases with time. Moreover, phosphate ions react with Fe ions released from the surface and form deposits preferentially at the surface defect sites. The deposits incorporate into the Mefp-1/ceria composite film and heal the surface defects, which result in a significantly improved corrosion inhibition effect for the Mefp-1/ceria composite film in both initial and prolonged exposure situationsQC 20130610
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Silva, Sueli Maria da. "Estudo da interação entre albumina do soro bovino (BSA) e nanopartículas de maghemita (Y-Fe2O3) funcionalizadas com três diferentes ligantes aniônicos." Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/7803.
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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Biological applications of nanoparticles require understanding the interaction between proteins and nanoparticles. In this thesis were performed studies of interaction between the protein BSA and maghemite nanoparticles functionalized with different anionic ligands (citrate ions, tripolyphosphate ions and bilayer laurate ions ), by three analytical techniques: isothermal titration calorimetry (ITC), adsorption isotherm by hydrodynamic diameter measurements using dynamic light scattering (DLS) and fluorescence spectroscopy. The values of the interaction constants (Ka) of BSA association on nanoparticles surface by DLS measurements were similar to those obtained by ITC, for the three systems. On the other hand, the study by ITC did not allows determining of the stoichiometry values (Nmax) of the association processes. The results obtained by the fluorescence technique are highly discrepant of the results obtained by the other two techniques. The evaluation of the functionalizing agent effect on the interaction between magnetite nanoparticles and BSA showed that there are differences in the Ka values, and in the energetic profiles of the interactions for the three systems studied. The Ka value for the interaction between BSA and citrate functionalized nanoparticles was in the order of 106 M-1, whereas for the other systems the values of Ka were 104 M-1. The energetic profile of the interactions, was endothermic in the system BSA-NP-Citrate, and exothermic for BSA-NP-Laurato and for BSA-NP-Tripolyphosphate. From the analysis of the thermodynamic parameters, it was possible to suggest that the interaction in the BSA-NP-citrate system was predominantly electrostatic, whereas the interaction in the other systems predominantly involved hydrogen bonds. The albumin estearase activity was reduced by the interaction with the nanoparticles, and was dependent upon the nanoparticles concentration. The reduction in stearase activity was higher for the -BSA-NP-Laurate system. In this work, the dynamic light scattering technique (DLS) was used, for the first time, to study of adsorption of BSA on functionalized maghemite nanoparticles. In addition, under the experimental conditions used, DLS was the only technique that provided (Nmax) values similar to those estimated.
Aplicações biológicas de nanopartículas inorgânicas demandam o entendimento das interações entre as nanopartículas e as proteínas. Nessa tese foram realizados estudos de interação entre a proteína BSA e nanopartículas de maghemita funcionalizadas com diferentes ligantes aniônicos (íons citrato, íons tripolifosfato e bicamada de íons laurato), por três técnicas analíticas: titulação de calorimetria isotérmica (ITC), isoterma de adsorção por medidas de diâmetro hidrodinâmico, empregando espalhamento de luz dinâmico (DLS) e espectroscopia de fluorescência. Os valores das constantes de interação (Ka) obtidos pelos estudos de adsorção da BSA sobre as nanopartículas por medidas de DLS foram semelhantes aos obtidos por ITC, para os três sistemas. Por outro lado, os estudos por ITC, não permitiram a determinação dos valores das estequiometrias de reação (Nmax). Os resultados obtidos pela técnica de fluorescência são altamente discrepantes em relação aos resultados obtidos pelas outras duas técnicas. A avaliação do papel do agente funcionalizante sobre a interação entre as nanopartículas e a BSA mostrou que há diferenças no perfil das interações nos três sistemas estudados. O valor da constante de associação para o sistema BSA-NP-Citrato foi da ordem de 106 mol.L-1, enquanto que para os demais sistemas foi de 104 mol.L-1. Os parâmetros termodinâmicos obtidos para o sistema BSA-NP-Citrato (ΔH = 5x103 cal.mol-1; ΔS+ +45 cal.mol-1; ΔG= -8410 cal.mol-1) sugerem que o processo de adsorção foi predominantemente de natureza eletrostática. Por outro lado, os parâmetros termodinâmicos obtidos para os sistemas, BSA-NP-Laurato (ΔH=-1,38x105 cal.mol-1; ΔS= -441cal.mol-1; ΔG= -6582 cal.mol-1) e BSA-NP-Tripolifosfato (ΔH = - 1,86x104 cal.mol-1; ΔS= -41 cal.mol-1; ΔG= -6352 cal.mol-1) sugerem que o processo de adsorção nesses sistemas tenha ocorrido predominantemente por pontes de hidrogênio. A atividade de esterase da albumina foi reduzida pela interação com as nanopartículas, e foi dependente da concentração das mesmas. A redução na atividade da esterase ocorreu em maior extensão para o sistema BSA-NP-Laurato. Nesse trabalho, a técnica de espalhamento de luz dinâmico (DLS) foi empregada pela primeira vez para o estudo de adsorção de BSA sobre nanopartículas de maghemita funcionalizadas, e se mostrou adequada. Além disso, nas condições experimentais utilizadas, foi a única técnica que forneceu valores da estequiometria de reação (Nmax) semelhante aos valores estimados.
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Dyne, Eric D. "Magnetic Nanoparticle Hyperthermia-Mediated Clearance of Beta-amyloid Plaques: Implications in the Treatment of Alzheimer’s Disease." Kent State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=kent1618706341759415.
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Lagoutte, Priscillia. "L'ingénierie protéique moderne : de l’évolution moléculaire dirigée à la conception rationnelle de biomolécules à intérêt diagnostique et vaccinal." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1160.
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L’ingénierie protéique servant autrefois à comprendre les relations structures-fonctions des protéines connait un tournant majeur depuis plusieurs années. L’ingénierie protéique évolue pour créer des nouvelles fonctions protéiques : c’est la naissance de l’ingénierie protéique moderne. L’objectif de ma thèse a consisté à mettre en place et caractériser deux approches indépendantes d’ingénierie protéique dans le domaine du vaccin et du diagnostic. Le premier projet consistait à générer des ligands protéiques à partir d‘échafaudages moléculaires (des alternatifs aux anticorps) en couplant le ribosome display au NGS et en développant des outils d’analyses bio-informatiques. Des sélections contre des cibles protéiques d’origine bactérienne et virale ont conduit à l’identification de ligands Affibodies affins (µM au nM). Leur caractérisation a validé leur potentiel comme outil de recherche et de réactif diagnostique. Ces études ont permis de valider la plateforme de génération des ligands mise en place, en augmentant l’exploration de l’espace de diversité des interactions des ligands. Le second projet portait sur le développement d’une plateforme de présentation et de vectorisation à partir de particules d’encapsuline. Elles ont été génétiquement modifiées pour présenter de manière répétée à leur surface l’ectodomaine de la protéine de matrice M2 (M2e) du virus Influenza A H1N1 tout en encapsulant une protéine hétérologue : l’eGFP. Les nanoparticules modifiées sont correctement formées et encapsulent l’eGFP. Des souris immunisées par ces particules induisent une réponse anticorps spécifique contre l’épitope M2e et l’eGFP. L’utilisation de ces nanoparticules comme plateforme vaccinale de présentation et de vectorisation est prometteuse et ouvre la voie pour d’autres applications en biotechnologieIn the past, protein engineering used to understand function and structure relationship. But since few years, protein engineering was used to create new protein functions: modern protein engineering was born. The aim of my thesis was to set up and characterize two approaches of protein engineering in diagnostic and vaccine field. The first project was to generate artificial binder using protein scaffolds as an alternative to antibodies by coupling ribosome display (RD) to NGS and developing bio-informatics tools. Screening and selection against bacterial and viral targets have led to affibody binder’s identification with an affinity range from µM to nM. Their characterization has validated their potential as research tools and protein reagents for diagnostic assay. Coupling ribosome display to high throughput sequencing as means to directly identify selected binder coding sequences, enormously enhance binder discovery depth. The second project was to generate an innovative nanocarrier based on encapsulin nanoparticle, for customized peptide display and cargo protein vectorization. Encapsulin particles from T.maritima were genetically modified for simultaneous display of the matrix protein 2 ectodomain of the influenza H1N1 A virus and heterologous protein eGFP packaging. Genetically engineered encapsulin nanoparticles were well-formed and abled to efficiently load eGFP. Immunogenicity studies revealed antibody responses against both the surface epitope and the loaded cargo protein. Taken together, this display system is a versatile tool for rational vaccine design and paves the way for new applications in the research fields of vaccine, antimicrobial research and other biotechnological applications
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Vincent, Abhilash. "Probing the Nanoscale Interaction Forces and Elastic Properties of Organic and Inorganic Materials Using Force-Distance (F-D) Spectroscopy." Doctoral diss., University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4251.
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Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 µN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.Ph.D.
Department of Mechanical, Materials and Aerospace Engineering;
Engineering and Computer Science
Materials Science & Engr PhD
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Jorge, Manuel Fernando Coronado. "Caracterização de filmes nanocompósitos biodegradáveis a base de gelatina produzidos com um aplicador automático de filmes." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/74/74131/tde-20112012-135548/.
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O desenvolvimento de filmes à base de biopolímeros tem sido bastante estudado devido aos problemas ambientais causados pelas embalagens de plásticos sintéticos. Dentre os biopolímeros, merece destaque a gelatina, que tem excelente propriedade filmogênica. Entretanto, até o presente momento, os filmes de gelatina apresentam limitações de propriedades mecânicas e, tem elevada sensibilidade à umidade. Uma alternativa para melhorar as propriedades desses filmes pode ser a utilização de nanopartículas como carga de reforço. Assim, os objetivos desta tese foram o desenvolvimento e a caracterização de filmes à base de gelatina, reforçados com nanopartículas. Especificamente, foram objetivos os seguintes estudos: avaliação do efeito da concentração de gelatina sobre as propriedades reológicas das soluções formadoras de filmes e de algumas propriedades físicas de filmes de gelatina, preparados com essas soluções formadoras de filmes com um aplicador automático de filmes; e estudo dos efeitos das concentrações de gelatina e de montmorilonita sobre as propriedades físicas de filmes nanocompósitos e de suas soluções formadoras. Em ambos os estudos, os filmes foram caracterizados imediatamente após a secagem e depois de 7 dias de acondicionamento. As soluções formadoras de filmes (SFF) foram preparadas com gelatina e glicerol, e as soluções formadoras de nanocompósitos (SFN), pela mistura de uma solução de gelatina com uma dispersão de montmorilonita em água, em ambos os estudos, em proporções e temperaturas convenientes. Após a homogeneização, as SFF ou SFN foram resfriadas até a temperatura adequada para aplicação em suportes de plexiglass com a ajuda de um espalhador automático de filmes, acoplado a um banho ultratermostatizado. A altura do espalhador foi mantida constante em 1,5 mm, e a velocidade de espalhamento foi fixada em 35 mm/segundo. Em seguida, as SFF ou SFN foram desidratadas a 30ºC por 24h, em estufa com circulação de ar. No primeiro estudo, as SFF foram preparadas com 5, 8, 11 e 14g de gelatina/100g de SFF e 30 g de glicerol/100g de gelatina, e na segunda parte da tese, as SFN foram preparadas com 5 e 8g de gelatina/100g de SFN, 30 g de glicerol/100g de gelatina e 0, 5, 10 e 15g de montmorilonita/100g de gelatina. As propriedades reológicas das SFF e SFN foram estudadas com testes estacionários e dinâmicos. As propriedades reológicas dasdispersões de montmorilonita em água também foram estudadas, bem como a determinação do tamanho médio das partículas e do potencial zeta. Os filmes foram caracterizados para conhecimento da espessura, umidade, propriedades mecânicas (testes de tração e perfuração), transições de fase, microestrutura das superfícies e da criofratura, cristalinidade, espectroscopia de infravermelho com transformada de Fourier (FTIR), hidrofobicidade, isoterma de sorção de vapor de água, solubilidade em água, permeabilidade ao vapor de água, ao O2 e CO2, cor, opacidade, brilho e propriedade de barreira a luz/UV, segundo os objetivos específicos de cada estudo. Em relação ao primeiro estudo, observou-se que a concentração de gelatina na SFF influenciou fortemente todas as propriedades reológicas e inclusive, as transições sol-gel e gel-sol, determinadas nos testes dinâmicos com varredura de temperatura. A concentração de gelatina na SFF aumentou linearmente a espessura dos filmes, sem, contudo afetar nitidamente a umidade, as transições de fases e nem as propriedades mecânicas obtidas nos testes de tração. A força na perfuração aumentou, mas esse efeito foi devido ao aumento da espessura dos filmes. Em relação ao estudo sobre nanocompósitos de gelatina, observou-se inicialmente que a montmorilonita dispersa em água apresentou diâmetro médio entre 204 e 344 nm, e potencial zeta variando em torno de -43mV. A carga de nanopartícula não afetou a viscosidade da dispersão em água, mas influenciou fortemente as propriedades reológicas e de transições de fases das SFN. A carga de nanopartículas também influenciou a espessura, as propriedades mecânicas e a hidrofobicidade dos filmes nanocompósitos, sem uma função nítida. Comportamento similar foi observado nos resultados da calorimetria diferencial de varredura, embora o padrão de cristalinidade, os espectros de FTIR e as microestruturas dos filmes tenham variado com a carga de montmorilonita. O filme nanocompósito produzido com 5g de gelatina/100g de SFN e 5g de montmorilonita/100g de gelatina apresentou melhores propriedades mecânicas e menor solubilidade em água. Esse filme foi submetido a caracterizações complementares com resultados típicos de filmes de gelatina. No geral, pode-se concluir que a montmorilonita pode melhorar as propriedades dos filmes de gelatina, porém, mais estudos serão necessários para se garantir perfeita dispersão da nanopartícula na matriz do filme.The development of films based on biopolymers has been widely studied due to the environmental problems caused by synthetic plastic packaging. Among biopolymers, gelatin has to be outlined as it has excellent filmogenic properties. However, until now, the gelatin films have mechanical properties limitations and, high humidity sensitive. An alternative to improve the properties of these films may be the use of nanoparticles as load. Thus, the objectives of this thesis were the development and characterization of gelatin-based films, charged with nanoparticles. Specifically, the objectives were the following studies: evaluation of the effect of gelatin concentration on the rheological properties of the film-forming solutions and on some physical properties of the gelatin films prepared with those film forming solutions using a automatic spreader; and the study of the effect of the gelatin and montmorillonite concentrations on the physical properties of nanocomposite films and their film forming solutions. In both studies, the films were characterized immediately after drying and then after 7 days of conditioning. The film forming solutions (FFS) were prepared with gelatin and glycerol, and the nanocomposite film forming solutions (NFS), by blending the gelatin solution with the montmorillonite dispersion in water, for both studies, in convenient proportions and temperatures. After homogenization, the FFS or NFS were cooled to a proper temperature and applied on plexiglass plates with the help of an automatic spreader, attached to an ultra thermostatized bath. The height of the spreader was kept constant at 1.5 mm, and the spreading speed fixed at 35 mm/second. After that, the FFS or NFS were dehydrated at 30ºC for 24h, in an oven with air circulation. In the first study, the FFS were prepared with 5, 8, 11 and 14g of gelatin/100g of FFS and 30g of glycerol/100g of gelatin, and in the second part of the thesis, the NFS were prepared with 5 and 8g of gelatin/100g of NFS, 30 g of glycerol/100g of gelatin and 0, 5, 10 and 15g of montmorillonite/100g of gelatin. The rheological properties of the FFS and NFS were studied by steady and dynamic tests. The rheological properties of the montmorillonite dispersions in water were also studied, along with the average particle size and zeta potential determinations. The films were characterized to determine thickness, water content, mechanical properties (tensile and puncture tests), phase transitions, surface and cryo-fracture microstructure, crystallinity, Fourier transform infrared spectroscopy (FTIR), hydrophobicity, water vapor sorption isotherm, solubility in water, water vapor, O2 and CO2 permeabilities, color, opacity, gloss, and light/UV barrier properties, according to the objectives of each study. Regarding the first study, it was observed that the gelatin concentration in the FFS strongly influenced the rheological properties and even the sol-gel and gel-sol transitions, determined by the dynamic temperature scanning tests. The gelatin concentration provoked linear increasing of films thickness without, however, affecting discernibly the water content, the phase transitions neither the mechanical properties determined by the tensile tests. The puncture force increased, but this effect was due to the thickness increasing of the films. Regarding the gelatin nanocomposites study, it was initially observed that the montmorillonite dispersed in water had average diameter between 204 and 344 nm, and zeta potential varying around -43mV. The nanoparticle filling did not affect the viscosity of the water dispersion, but strongly influenced the rheological properties and the phase transitions of the NFS. The nanoparticle filling also affected the thickness, the mechanical properties and the hydrophobicity of the films, without a clear function. Similar behavior was observed for the differential scanning calorimetry results, although the crystalline pattern, the FTIR spectra and the microstructure of the films varied with the montmorillonite filling. The nanocomposite film produced with 5g of gelatin/100g of NFS and 5g of montmorillonite/100g of gelatin showed better mechanical properties and lower solubility in water. That film was submitted to further characterizations rendering results typical of gelatin films. In general, it can be concluded that the montmorillonite can improve the properties of gelatin films, however more studies will be necessary to guarantee the perfect dispersion of the nanoparticles within the film matrix.
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Tahir, Mukarram Ahmad. "Protein mimetic nanoparticles." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/121606.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2019Cataloged from PDF version of thesis.
Includes bibliographical references (pages [121]-140).
Gold nanoparticles with amphiphilic surface functionalization have been shown to spontaneously fuse with lipid bilayers through a non-endocytic mechanism that generates minimal membrane perturbation. The membrane translocation capability of these nanoparticles makes them attractive candidates for engineering clinical applications that operate on a single-cell resolution. In particular, the physiochemical similarity between these nanoparticles and membrane-bound and free-circulating proteins suggests a possibility for designing nanostructures that can function as synthetic alternatives to proteins. In this thesis, we demonstrate how molecular simulation techniques have allowed us to tackle this engineering challenge and develop nanoparticles that can modulate fusion between lipid membranes, transport hydrophobic small molecules to lipid-bound compartments, and modify the permeability of lipid membranes. These are concrete realizations of nanoparticles functioning as protein mimics, and unlock new avenues of research on how nanomaterials can be designed from first principles to perform targeted functions in biological systems.
by Mukarram Ahmad Tahir.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Materials Science and Engineering
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Barbero, Francesco. "Physicochemical characterization of the evolution of metal nanoparticles in biological and environmental media: from synthesis to interaction with living organisms." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/670187.
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La producció creixent de Nanopartícules (NPs) conduirà inevitablement a un augment d’exposició humana i ambiental a aquests materials. En conseqüència, han sorgit preocupacions raonables sobre els seus possibles riscos per a la seguretat, donant lloc a la disciplina de nanotoxicologia/nanoseguretat.
A causa de l’alta reactivitat, les NP exposades a diferents escenaris biològics i ambientals, solen arribar a un estat termodinàmic més estable mitjançant l’agregació, la interacció amb les molècules presents al medi, l’adsorció a matèria macro-orgànica, les transformacions químiques i la dissolució. Totes aquestes transformacions poden generar una nova identitat dels nanoobjectes o produir noves entitats químiques, canviant així el seu comportament i, per tant, el risc potencial associat. Així, els mateixos NP poden tenir un destí totalment diferent i, en conseqüència, un impacte totalment diferent sobre els organismes vius i el medi ambient depenent del microambient (per exemple, el medi d’exposició) on es trobin. A més, les característiques primigènies del nanomaterial influeixen molt en la seva destinació biològica i ambiental. Des d’aquesta perspectiva, és fonamental comprendre les característiques de l’objecte final que toparà amb els organismes vius i analitzar les seves propietats, per tal de correlacionar les característiques prèvies i finals de NP amb els efectes potencials sobre els organismes vius.
En aquest context, aquesta tesi s’ha centrat en la transformació fisicoquímica dels NP model exposats a medis biològics i ambientals. Per a aquests estudis, es van triar NPs Au i Ag, ja que són models NP molt utilitzats i per les seves nombroses aplicacions. En primer lloc, l’estudi es va centrar en la influència de la composició de medis de cultiu cel·lular en el procés de formació de protein corona (PC), la composició final i l’estat d’agregació de NPs i els efectes consegüents sobre la captació de cèl·lules NP. També es va realitzar una caracterització fisicoquímica de la naturalesa de la bicapa CTAB - Au NP Au per estudiar l’impacte d’aquest recobriment de superfície NP àmpliament utilitzat en l’exposició de la partícula a fluids biològics, en la formació de la proteïna corona i en el disseny i interpretació de Proves de toxicitat NP. Finalment, es va explorar l’evolució del NP en aigua dolça natural mitjançant un estudi de la naturalesa d’interacció de les NP i de la matèria orgànica natural i de les característiques derivades del NP.La creciente producción de nanopartículas (NP) conducirá inevitablemente a un aumento de la exposición humana y ambiental a estos materiales. En consecuencia, han surgido preocupaciones razonables con respecto a sus posibles riesgos de seguridad, dando lugar a la disciplina de nanotoxicología/nanoseguridad. Debido a la alta reactividad, los NP expuestos a diferentes escenarios biológicos y ambientales tienden a alcanzar un estado termodinámico más estable a través de la agregación, la interacción con las moléculas presentes en el medio ambiente, la adsorción a la materia macro-orgánica, las transformaciones químicas y la disolución. Todas estas transformaciones pueden generar una nueva identidad de los nanoobjetos o producir nuevas entidades químicas, cambiando así su comportamiento y, en consecuencia, su riesgo asociado potencial. Por lo tanto, los mismos NP pueden tener un destino totalmente diferente y, en consecuencia, un impacto totalmente diferente en los organismos vivos y el medio ambiente dependiendo del microambiente (por ejemplo, el medio de exposición) en el que se encuentran. Además, las características prístinas del nanomaterial influyen mucho en su destino biológico y medioambiental. Desde esta perspectiva, resulta fundamental comprender las características del objeto final que encontrará organismos vivos y analizar sus propiedades, a fin de correlacionar las características de NP prístinas y finales con los posibles efectos sobre los organismos vivos. En este contexto, el objetivo de esta tesis ha sido el estudio de la transformacion fisicoquímico de NP modelo expuestos a medios biológicos y ambientales. Para estos estudios, se eligieron NPs de Au y Ag, ya que son modelos de NP ampliamente utilizados y debido a sus numerosas aplicaciones. En primer lugar, el estudio se centró en la influencia de la composición de los medios de cultivo celular en el proceso de formación de protein corona, la composición final y el estado de agregación de NP y los efectos consiguientes en la absorción de células NP. También se realizó una caracterización fisicoquímica de la naturaleza de la bicapa CTAB - Au NP para estudiar el impacto de este recubrimiento de superficie NP ampliamente utilizado en la exposición de la partícula a los fluidos biológicos, en la formación de la corona de proteínas y en el diseño e interpretación de Pruebas de toxicidad NP. Finalmente, la evolución de NP en agua dulce natural se exploró mediante la realización de un estudio de la naturaleza de interacción de NP y materia orgánica natural y las características derivadas de NP.
The increasing production of engineered Nanoparticles (NPs) will inevitably lead to an increase of human and environmental exposition to these materials. Consequently reasonable concerns have arisen regarding their potential safety risks, giving rise to the nanotoxicology/nanosafety discipline. Because of the high reactivity, NPs exposed to different biological and environmental scenarios, tend to reach a more stable thermodynamic state via aggregation, interaction with the molecules present in the environment, adsorption to macro-organic matter, chemical transformations and dissolution. All these transformations can generate a new identity of the nano-objects or produce new chemical entities, thereby changing their behaviour and consequently their potential associated risk. Thus, the same NPs can have a totally different fate and consequently a totally different impact on living organisms and the environment depending on the microenvironment (e.g., the exposure medium) in which they are. Furthermore, the pristine features of nano-material highly influence their biological and environmental fate. From this perspective, it becomes fundamental to understand the characteristics of the final object that will encounter living organisms and analyze its properties, in order to correlate the pristine and final NP features with the potential effects on living organisms. In this context, the focus of this thesis has been on the physicochemical transformation of model NPs exposed to biological and environmental media. For these studies, Au and Ag NPs were chosen as they are widely used NP models and because of their numerous applications. Firstly, the study focused on the influence of the cell culture media composition on the protein corona (PC) formation process, final composition and NPs aggregation state and the consequent effects on NP cell uptake. A physicochemical characterization of the nature of the CTAB - Au NP bilayer was also carried out to study the impact of this widely used NP surface coating on the particle’s exposition to biological fluids, on the formation of the protein corona and on the design and interpretation of NP toxicity tests. Finally, the NP evolution in natural fresh water was explored by carrying out a study of the interaction nature of NPs and natural organic matter and the deriving NP features.
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Gauche, Cony. "Synthèse et études de l'auto-assemblage en solution de diblocs amphiphiles à base de xyloglucanes et application pour la stabilisation de protéines." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00949397.
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Ce travail décrit une nouvelle route synthétique qui a pour objectif l'obtention de diblocs amphiphiles constitués uniquement d'oligosaccharides issus de xyloglucanes des graines de Tamarin. Les xylogluco-oligosaccharides (XGOs, DP7, 8, 9) de tailles parfaitement définies ont été obtenus par une digestion enzymatique contrôlée (cellulase) de xyloglucanes. Dans la perspective de lier les deux blocs par cycloaddition 1,3-dipolaire de Huisgen catalysée par le Cuivre I, dite aussi chimie " click ", les XGOs ont subit une réaction d'amination réductrice assistée par micro-ondes. L'action de la propargylamine a permis d'intégrer en position réductrice du XGO la fonction alcyne et une peracétylation des focntions hydroxyles du sucre ont rendu ce bloc hydrophobe. D'un autre côté, l'azidoethylamine a permis d'insérer la fonction azoture et constitue le bloc hydrophile. Cette stratégie de synthèse a également été transposée à un oligosaccharide monodisperse (XGO, DP7) provenant de la dégalactosylation enzymatique du xyloglucane par l'action supplémentaire de la galactosidase d'Aspergillus Niger. Finalement, les diblocs amphiphilies ont été synthétisé aussi bien à partir des XGOs de DP7, 8, 9 (XGO-b-XGO,Ac), que du XGO DP7 (DP7-b-DP7,Ac). Leurs propriétés d'auto-assemblages dans l'eau ont été réalisées ainsi que leur caractérisation physico-chimique. Suite à des mesures de concentration micellaire critique (CMC) obtenus par spectroscopie de fluorescence du pyrène, nous avons observé que l'élimination des unités de galactose provoque une augmentation de la CMC. La détermination du diamètre des micelles en solution aqueuse a été réalisée grâce à la technique de diffusion de la lumière (DLS) et a été confirmée par microscopie électronique à transmission (MET). Des micelles sphériques d'une taille moyenne de 25 nm (XGO-b-XGO,Ac) et de 6 nm (DP7-b-DP7,Ac) ont été observées au MET. La digestion enzymatique partielle des micelles formés à partir du dibloc XGO-b-XGO,Ac dans l'eau, conduisant à la formation des micelles DP7-b-XGO,Ac a conduit à un système moins polydisperse et à une diminution de la taille moyenne du diamètre micellaire de l'ordre de 50% (déterminée par DLS). Des nanoparticules de gliadine et de zéine ont été préparées par désolvatation en utilisant le dibloc XGO-b-XGO,Ac comme surfactant en comparaison au surfactant commercial non-ionique, le Pluronic F68. Les résultats suggèrent la capacité du dibloc à stabiliser la protéine de zéine sous forme de nanoparticules sphériques et de façon relativement monodisperses. Les particules formées et stabilisées grâce à l'association de protéines d'origine végétale et d'un surfactant " biopolymérique " synthétisé uniquement par des oligosaccharides, apparaissent comme des systèmes idéaux, associant biocompatibilité, biodégradabilité et des origines naturelles et renouvelables. Ces systèmes peuvent tout à fait être valorisés pour la libération contrôlée de substances actives.
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Pollitt, Michael John. "Protein coating of nanoparticles." Thesis, University College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442065.
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Soumbo, Marvine. "Adsorption des protéines sur les surfaces de couches minces de silice seules ou additivées de nanoparticules d'argent : impact sur les forces d'adhésion de Candida albicans." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30258.
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Dans de nombreux secteurs, l'adhésion microbienne sur les surfaces est la source de multiples impacts négatifs. Cette étape est considérée comme préliminaire au développement de biofilm et peut être influencée par la présence d'un film conditionnant engendré par l'adsorption des protéines sur la surface. Ainsi, les stratégies visant une intervention au moment de la phase initiale d'adhésion représentent une approche appropriée pour prévenir la bio-contamination des surfaces et nécessitent une compréhension à l'échelle moléculaire. Dans ce contexte, les matériaux nanocomposites à base de nanoparticules d'argents (AgNPs) et de silice (SiO2) apparaissent comme des outils pertinents. Ce travail de thèse porte sur l'utilisation de substrats nanocomposites possédant une monocouche d'AgNPs exposées à leurs surfaces ou enterrées dans une matrice de SiO2plasma à une distance contrôlée de quelques nanomètres de la surface afin d'explorer, d'une part l'adhésion de protéines modèles (Sérum Albumine Bovine, DsRed et Fibronectine) et leurs changements conformationnels et d'autre part, la cinétique de détachement de la levure Candida albicans dans les différentes conditions. Les AgNPs sont bien connues pour leurs activités antimicrobiennes et présentent de plus, des propriétés optiques permettant de détecter des signatures moléculaires à leurs proximités. Suite à l'application de la spectroscopie Raman exaltée de surface en utilisant les couches nanocomposites à base d'AgNPs, la détection de trois conformations de la DsRed (protéine fluorescente rouge) adsorbée et déshydratée sur les substrats plasmoniques a été possible. Les résultats obtenus montrent que les changements conformationnels des protéines avec une forte cohérence interne sont réversibles. En parallèle, nous avons évalué la dynamique d'organisation et le comportement de la SAB, de la Fn et de la DsRed en contact avec des couches minces de silice ou additivées d'AgNPs. Les mesures des angles de contact des gouttelettes de différentes concentrations protéiques ont montré une interaction hydrophile croissante avec la SiO2th thermique. L'hydrophobicité de surface est modifiée pour les substrats nanocomposites. L'épaisseur et les propriétés optiques des couches protéiques adsorbées ont été évaluées par ellipsométrie spectroscopique. En fonction de la concentration de protéines dans solution les résultats montrent l'évolution d'une monocouche protéique non continue et non dense vers une monocouche plus compacte et plus complexe pour des concentrations élevées.[...]Microbial adhesion on solid surfaces is the source of multiple negative impacts in many areas. This step is considered prior to biofilm formation. It might be influenced by the presence of a conditioning layer generated after protein adsorption on the surface. Thus, strategies to act during the initial phase of microbial adhesion represent an appropriate approach to prevent bio-contamination of solid surfaces. However, they require understanding of the underlying mechanisms at the molecular level. In this context, nanocomposite materials based on silver nanoparticles (AgNPs) and silica (SiO2) appear as relevant tools. This thesis focuses on the use of nanocomposite thin layers containing a plan of AgNPs exposed on their surfaces or buried in a SiO2plasma matrix at a controlled distance of a few nanometers from the surface in order to explore, on the one hand, the adhesion of model proteins (Bovine Serum Albumin, DsRed and Fibronectin) and their conformational changes and secondly, the kinetics of detachment of the yeast Candida albicans under the different conditions. AgNPs are well known for their antimicrobial activities but also for their optical properties allowing detection of molecular signatures at their proximities. Following the application of surface-enhanced Raman spectroscopy using AgNP-based nanocomposite layers, the detection of three conformations of DsRed (red fluorescent protein) adsorbed and dehydrated on plasmonic substrates was achieved. The obtained results show that the conformational changes of proteins with a strong internal coherence are reversible. In parallel, we have evaluated the dynamics of the organization and behavior of BSA, Fn and DsRed in contact with thin silica layers or silica layers containing AgNPs. Contact angle measurements of droplets of different protein concentrations showed increasing hydrophilic interaction with thermal SiO2th. For the nanocomposite layers, the surface hydrophobicity is modified. The thickness and optical properties of the adsorbed protein layers were evaluated by spectroscopic ellipsometry. Depending on the protein concentration in solution the results show the evolution of a non-continuous and non-dense protein monolayer to a more compact and complex monolayer at high concentrations. [...]
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Ma, Wenwei. "Protein-nanoparticles interaction and assembly." Thesis, University of Lincoln, 2017. http://eprints.lincoln.ac.uk/30884/.
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Nanoparticles are increasingly important in biotechnology as they are extensively used as drug delivery carriers and in biosensors. In both these two contexts, protein-nanoparticle interactions are often involved. Proteins that are present in body fluids inevitably interact with nanoparticle based drug carriers and typically surround them forming the so called “protein corona”. Biosensors that are based on nanoparticles often have proteins deliberately attached to their surface, for example antibodies that bind specific analytes. The understanding of the assembly mechanisms at the protein-nanoparticle interface and the ability to engineer proteins that interact with nanoparticles in the desired way, are therefore two essential requisites for the future development of nano-medicines and nano-biosensors. In this work, we focused on the interaction of proteins with gold nanoparticles (GNPs). GNPs are available with a broad range of surface chemistries, suitable for the conjugation of many biomolecules. Although there are at least three decades of studies on gold colloids with different surface chemistries, there is still quite little known about what are the exact features of a protein that determine its adsorption onto gold. We developed methods to study this and applied them to characterise the adsorption on GNPs of Glutathione-S-Transerase (GST), which was reported previously as a protein that strongly binds gold. We determined its affinity and kinetics of binding and unravelled the mechanism of its thiol-mediated chemisorption. We found that GST binds to GNPs even more efficiently than other known gold-binding proteins, such as Bovine Serum Albumin (BSA). We concluded that GST could be considered a very useful gold-protein interface, especially considering that GST fusion is routinely used for affinity purification of recombinant proteins and therefore well established. We also fused self-assembling proteins to GST or chemically cross-linked them to BSA. The scope was to explore the feasibility of hierarchical and ordered assembly of designer proteins onto GNPs, with the ultimate goal of providing a convenient tool for modular assembly of proteins onto nanomaterials. It is known that proteins tend to denature and lose their function when in contact with GNPs, which is not optimal for biosensors or in nanomedicine. We found that it is possible to use GST or BSA to form a sacrificial layer on gold, which exposes linked, self-assembling proteins that are able to bind their counterpart, unaffected by the GNP surface. We reported two proof-of-concepts: the first based on mimics of the self-assembling neuronal SNARE proteins and the second based on the pair SpyCatcher/SpyTag, derived from Streptococcus pyogenes proteins and used in bio-conjugation for their ability to self-catalyse the formation of isopeptidic bonds. We believe that the novel methods and original results presented in this thesis apply to both the understanding and the engineering of the protein-nanoparticle interface and will be beneficial for the broad nanobiotechnology community. In fact, our findings have potential applications in a broad range of fields, spanning from the improvement of the circulation life-time of nanomedicines by preventing the binding of serum protein and opsonisation, to the improvement of the manufacturing of GNPs-based immune-biosensors such as those used in lateral flow devices.
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Maddala, Sai P. "Synthesis of phosphonate functionalized silica nanoparticles for protein immobilization, intracellular protein delivery and catalytic applications." Thesis, Queen Mary, University of London, 2014. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8961.
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Organosilica nanoparticles have attracted a lot of research interest in a variety of areas such as drug delivery and catalysis because of their properties which include high surface area as well as tunable particle and pore size. In particular, nanoparticles with large pore sizes are of great interest because of their potential to host large guest molecules such as proteins and as catalysts. The focus of the work in the thesis was to develop phosphonate functionalized organosilica nanoparticles for biomedical and catalytic applications. Raspberry textured phosphonatesilica nanoparticles denoted, RNPPME(2.5) (where the number in the brackets represents the moles of organophosphonate per gram), with large pore size (11–17 nm), uniform particle size (70 – 90 nm) and high surface area were produced through the use of template directed base catalysed synthesis, using tetraethylorthosilicate (TEOS) and dimethylphosphonatoethyltrimethoxysilane (DMPTMS) as the silica sources. The role of the reaction conditions such as temperature, surfactant concentration, pH, organosilane concentration and type were investigated and a mechanism for the raspberry nanoparticle formation was proposed. The particles were characterized using electron microscopy (SEM and TEM), Dynamic light scattering (DLS), silicon and phosphorus solid state NMR, and solution phase proton NMR of base digested particles, FT–IR, nitrogen adsorption porosimetry and thermal analysis (TGA). The ability of the particles to host protein molecules of the model protein, bovine serum albumin (BSA) was investigated and the particle–protein composite was characterized using circular dichroism (CD). Raspberry textured nanoparticles were found to host large quantities (26 wt%) of protein. Studies on other (small pore (3 nm) phosphonate functionalized nanoparticles NP_PME(0.2) and NP_PME(1.0)) and (3 nm pore) unfunctionalized mesoporous silica nanoparticles (MSN) revealed that phosphonate loading and the pore size influenced the protein uptake In addition to high protein uptake, the RNP_PME(2.5) particles also absorbed protein molecules rapidly (~ 20 minutes to maximum load). CD studies determined that the particle bound protein structure was not affected at physiological pH (7.40). The vast majority of the previously reported studies involving protein immobilization involved the use of bulk silica materials, which cannot be dispersed and hence those materials were unsuitable for in vivo protein delivery applications. The BSA@RNP_PME(2.5) particles showed good protein load and dispersion properties and hence are excellent protein delivery agents. Dispersions of nanoparticle composite BSA#@RNP*_PME(2.5) (where BSA# represents fluorescein isothiocyanate labelled BSA and RNP*_PME(2.5) represents rhodamine B isothiocyanate labelled RNP_PME(2.5)) was used to successfully deliver membrane impermeable protein BSA into HeLa cells. Intracellular protein delivery has attracted great interest due to its potential therapeutic applications and research tool value (e.g. for studying various cellular pathways). The toxicity of the guest free particles RNP*_PME(2.5) and the protein loaded particles BSA#@RNP*_PME(2.5) was studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The particles and the protein-particle composite were found to be non-toxic. The mechanism of the particle uptake by the cells was also studied. The unloaded (protein free) particles were found to be taken up by caveolar endocytosis pathway and the protein loaded particles were taken up by folic acid mediated pathway. The results indicated that the particles can successfully deliver membrane impermeable protein across the cell membrane. This result suggested that the particles can potentially be used for intracellular protein delivery applications. Raspberry textured nanoparticles RNP_PME(2.5) were also used to host the enzyme lipase. It was demonstrated that immobilization increased the maximum velocity and Michaelis constant of the enzyme and also that the particles offered protection against the denaturing agent, urea. Finally, in a chemical catalysis application, the RNP_PME(2.5) particles were used to synthesize the platform chemical HMF, through Brönsted acid catalysed dehydration of fructose. High yields of HMF (87 %) were achieved when 10 wt% fructose was used. The particles demonstrated good recyclability and also the ability to convert up to 50 wt% fructose into HMF (80 % yield). The particles therefore acted as protective agents for enzymes and can therefore be used as enzyme immobilizing agents. Additionally, they also acted as excellent Brönsted acid catalysts.
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Abu, Yazid Noraziah. "Proteases from protein-rich waste: production by SSF, downstream, immobilisation onto nanoparticles and application on protein hydrolysis." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405257.
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El trabajo realizado en esta tesis se enmarca dentro de las principales líneas de investigación del grupo de compostaje (GICOM), por una parte, en la valorización de residuos sólidos por medio de la fermentación en estado sólido (FES) para la obtención de productos de valor añadido en consoncia con el nuevo paradigma de considerar los residuos como materias primas y, en segundo lugar, en la utilización de nanomateriales para potenciar el uso de los productos obtenidos mediante FES.
La primera parte se centra en el aprovechamiento de residuos sólidos para la producción de enzimas de interés, concretamente proteasas. Las enzimas se produjeron a partir de la FES de la mezcla de residuo de pelo vacuno procedente de la industria de curtidos y lodos procedentes del proceso de digestión anaerobia utilizado como cosubstrato e inóculo. Por una parte se ha demostrado la capacidad de producir proteasas y sus posibles aplicaciones en la propia industria, ya que se han obtenido buenos resultados en el proceso del depilado del cuero. Además, se ha establecido un esquema para el tratamiento posterior a la fermentación, incluida la reutilización de los residuos generados a lo largo del proceso de modo que se aproxime a un sistema de residuo cero.
La segunda parte del trabajo consistió en una exploración preliminar para determinar cuáles son los soportes más eficientes para inmovilizar las proteasas producidas por FES de cara a su uso continuado y su mejor preservación. Se utilizaron proteasas obtenidas a partir de la FES de residuos ricos en nitrógeno, pelo vacuno utilizado en la primera parte del trabajo y residuo de soja de una industrial alimentaria. Se evaluó la aplicabilidad, reutilización, reproducibilidad y coste de los soportes para su uso en la inmovilización de dichas proteasas. La evaluación se efectuó con 10 materiales considerando su bio-compatibilidad con las proteasas: nanopartículas de oro (NpAu), perlas de quitosano (húmedas y secas), perlas de quitosano con nanopartículas de oro (Quitosan/Au), zeolitas funcionalizadas, resina aniónica A520, resina aniónica con nanopartículas de oro (A520/Au), nanopartículas de óxido de hierro (Fe3O4) y nanopartículas de óxido de titanio (TiO2). La eficiencia de la inmovilización se determinó a partir del rendimiento de inmovilización y la carga enzimática de cada soporte (U/mg). Las nanopartículas de óxido de hierro resultaron ser un soporte prometedor gracias a su bajo coste y a su fácil separación de forma magnética, aumentado así sus posibilidades de reutilización.
En último lugar, tanto las proteasas obtenidas a partir de los residuos de pelo como las de residuo de soja se inmovilizaron para ser evaluadas en la hidrólisis de proteínas, cuya eficiencia se comparó con la de las enzimas libres en el medio. Se utilizaron substratos de diferente origen (animales y vegetales): caseína, albúmina de huevo y proteína de salvado de avena, observado un efecto diferente según el origen de la proteasa utilizada. Estos resultados se corroboraron con los aminoácidos liberados tras la hidrólisis de las proteínas.
En términos globales, este estudio representa una investigación multidisciplinaria que abarca, por una parte, la gestión y valorización de residuos orgánicos mediante el proceso de fermentación en estado sólido, con la producción y recuperación del producto de interés, incluyendo la etapa de postratamiento y así como la aplicabilidad del producto. Además, la immbolización de las proteasas sobre nanopartículas de bajo coste se ha demostrado efectiva en la hidrólisis de proteinas tipo, consistuyendo una alternativa de bajo coste (tanto su producción como el soporte) a las técnicas actuales.In line with the main research lines of Composting research group (GICOM), this thesis focuses on the valorisation of solid organic residues and the utilisation of nanomaterials. Thus the thesis mutually linked the studies by transforming the solid waste using solid state fermentation (SSF) into value-added products and the use of nanoparticles to enhance the use of products obtained from SSF. The first major part covers the valorisation of solid waste and transforms it into valuable product namely protease. The protease was produced through the fermentation of hair waste and anaerobically digested sludge (ADS) acting as co-substrate and inoculum. For industrial application, the protease produced from this work has shown a good result on dehairing of cowhides. This chapter not only demonstrated from the production of protease until application on industrial activity but also exhibited the downstream processing in SSF and reutilisation of the remaining residues after SSF which was a crucial thing to establish a zero discharge. The second major part consisted of a preliminary screening work intending to determine the most suitable support for immobilisation of proteases. Proteases produced from two different protein-rich solid wastes: hair waste and soy fibre residue. The work aimed to find the feasible, reusable, reproducible, and cost-effective supports or carrier to be exploited for protease immobilisation. There were 10 materials were assessed for their biocompatibility with the protein: gold nanoparticles (AuNps), chitosan beads (wet and dry), chitosan beads coupled with gold nanoparticles (chitosan/Au), functionalised zeolite particles, the anionic resin (A520), anionic resin coupled with gold nanoparticles (A520/Au), magnetic iron oxide (Fe3O4) nanoparticles (MNPs), titanium oxide nanoparticles (TiO2_A and TiO2_B). The immobilisation efficiency was monitored based on immobilisation yield (% IY) and enzyme loading per carrier (U/mg). Iron oxide nanoparticles were shown as a promising support thanks to its low-cost and easy separation by a magnetic force, thus increasing their possibilities of reuse. In the last part of the thesis, both of the proteases from hair waste and soy fibre residue were immobilised on the support and were tested for application in protein hydrolysis. The efficiency of immobilised enzymes was compared with the free enzymes during the protein hydrolysis. Not to mention, the use of the different type of proteases (animal and plant origin) also was assessed during the protein hydrolysis of different type of protein (casein, egg white albumin, and oat bran protein isolate), since different protease produce different effect towards certain substrates. The amino acids released after hydrolysis reaction were well balanced with the degree of hydrolysis according to each protein substrates. Overall, the study represented a multidisciplinary research field spanning waste management using solid state fermentation including the downstream processing and its application. In addition, the immobilisation of proteases on low-cost nanoparticles has been shown to be effective in the hydrolysis of proteins, being a low-cost alternative (both production and support) to actual techniques.
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Chézeau, Laëtitia. "Étude au niveau pulmonaire du profil d’expression de gènes et de protéines chez le rat exposé par inhalation à un aérosol de particules nanostructurées de dioxyde de titane." Thesis, Université de Lorraine, 2018. http://www.theses.fr/2018LORR0226/document.
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En raison de l'utilisation croissante de nanomatériaux dans divers procédés industriels, le nombre de salariés potentiellement exposés ne cesse d’augmenter, sans que pour autant les propriétés toxicologiques de ces agents chimiques ne soient parfaitement connues. Comme des nanoparticules (NP) peuvent être mises en suspension dans les environnements de travail, l'inhalation représente la principale voie d'exposition professionnelle. Ainsi, l’évaluation des dangers associés à l’exposition à des aérosols nanostructurés nécessite de réaliser des études de toxicologie expérimentale par inhalation, en utilisant des modèles animaux. Dans ce travail, les propriétés toxicologiques pulmonaires d’un aérosol nanostructuré de dioxyde de titane (TiO2) ont été étudiées à court et long termes, en combinant des analyses toxicologiques conventionnelles (analyses du lavage broncho-alvéolaire (LBA), histopathologie des poumons et des ganglions lymphatiques); et de criblage moléculaire à haut contenu (analyses de transcriptomique et de protéomique). Des rats Fischer 344 ont été exposés par inhalation oro-nasale, à un aérosol nanostructuré de TiO2 à 10 mg / m3 ; 6 heures par jour, 5 jours par semaine pendant 4 semaines. Des échantillons biologiques ont été prélevés immédiatement et jusqu'à 180 jours suivant la fin de l'exposition. L'exposition à l'aérsosol nanostructuré de TiO2 a entraîné une importante réponse inflammatoire pulmonaire aiguë. Cette réponse était caractérisée par un influx de granulocytes neutrophiles, la présence de macrophages chargés en particules au niveau alvéolaire, la surexpression de gènes et de protéines impliqués dans les réponses inflammatoires et immunitaires, les cascades du complément et de la coagulation, le stress oxydant. Certains gènes surexprimés étaient également impliqués dans les lésions de l'ADN et la fibrose; et certaines protéines surexprimées étaient associées au protéasome et à l'organisation du cytosquelette. Dans le surnageant du LBA, l’augmentation du niveau d'histones et d'autres protéines associées aux pièges extracellulaires des neutrophiles (Neutrophilic Extracellular Trap, NET) suggère la libération de ces pièges extracellulaires dans l'espace alvéolaire. Cette libération possible de NET se produit dans un contexte inflammatoire mais en l'absence de changements histopathologiques significatifs. Ce processus inattendu n’a fait l’objet que de très peu d’études en lien avec une exposition à des nanomatériaux. Six mois après la fin de l'exposition (réponse à long terme), l'inflammation a diminué et s’accompagnait d’une baisse de la charge pulmonaire de titane (un marqueur fiable de la charge pulmonaire en nanoparticules de TiO2), mais de nombreux gènes et protéines étaient différentiellemment exprimés. Les conséquences physiopathologiques des changements rapportés ici ne sont pas entièrement connues, mais ces résultats devraient susciter des interrogations quant aux effets pulmonaires à long terme des NP inhalées biopersistantes de faible toxicité comme le TiO2. En conclusion, ce travail montre qu'il existe une bonne relation entre les changements cytologiques et histopathologiques d'une part et les modifications des profils d'expression de gènes et de protéines d'autre part. Cependant, dans certains cas, la transcriptomique et la protéomique pourraient être plus sensibles que les méthodes conventionnelles pour identifier de nouvelles propriétés toxicologiques, ou pour mieux comprendre les mécanismes moléculaires sous-jacents de la toxicité des produits chimiques. Notre étude avec d'autres travaux de la littérature pourraient également être utiles pour identifier des biomarqueurs d’exposition aux nanomatériaux ou prédire leurs effets nocifs à long termeDue to the growing use of nanomaterials in various industrial processes, the number of workers potentially exposed is increasing even though the toxicological properties of these compounds are not completely known. Since nanoparticles (NP) may get aerosolized, inhalation represents their main route of occupational exposure. Then, inhalation studies of nanomaterial toxicity in animal models appear to be the most relevant approach to assess their hazards. In this work, we studied the short and long term pulmonary toxicological properties of inhaled titanium dioxide (TiO2) nanostructured aerosol (NSA), using conventional (broncho-alveolar lavage (BAL) analyses, lung and lymph nodes histopathology); and high content molecular toxicological approaches (transcriptomics and proteomics analyses). Fischer 344 rats were exposed to 10 mg/m3 of TiO2 nanostructured aerosol by nose-only inhalation, 6h/day, 5 days/week for 4 weeks. Biological samples were collected immediately and up to 180 post-exposure days. Exposure to TiO2 NSA resulted in a strong acute pulmonary inflammation. This response was characterized by a neutrophil influx, the presence of particle-laden macrophages in the alveolar lumen, as well as overexpression of genes and proteins involved in inflammatory and immune responses, complement and coagulation cascades, oxidative stress. Some overexpressed genes were also involved in DNA damage and fibrosis; and some overexpressed proteins in proteasome and cytoskeleton organization. In the BAL supernatant, the increased level of histones and other neutrophilic extracellular trap (NET) -associated proteins suggests the release of these traps in the alveolar space. This possible NET release occurs in an inflammatory context but in the absence of significant histopathological changes. Very few studies reported this unexpected process related to exposure to nanomaterials. Six months after the end of exposure (long-term response), inflammation had decreased in line with the decrease of titanium lung burden (a surrogate for TiO2 pulmonary deposition), but many genes and proteins remained differentially expressed. The physiopathological consequences of the molecular changes reported here are not fully known, but these results should raise concern about the long-term pulmonary effects of inhaled low toxicity NP such as TiO2. Altogether, this work shows that there is a good relationship between cytological and histopathological changes in one hand and gene as well as protein expression profile modifications in the other hand. However, in some cases transcriptomics or proteomics could be more sensitive than conventional methods to identify new toxicological properties or to better understand the underlying molecular mechanisms of chemicals toxicity. Our study along with others could also be helpful to identify biomarkers of exposure or predict the long-term adverse effects of nanomaterials