L’objectif poursuivi au cours de ce travail est l’encapsulation de complexes métalliques au sein de nanoparticules biocompatibles, et ce pour des visées diagnostiques. Dans ce but, un protocole de double émulsion-diffusion de solvant Wi/O/We, n’utilisant que des composés biocompatibles, a été mis au point et optimisé pour obtenir, de façon quantitative et reproductible, des nanoparticules de PLGA de diamètre compatible avec une injection par voie parentérale. Cette formulation a été employée avec succès pour l’encapsulation de complexes modèles de Cu(II). Les formulations optimales permettent d’obtenir des nanoparticules possédant des diamètres hydrodynamiques moyens inférieurs à 200 nm avec des efficacités d’encapsulation entre 20 et 25 %. L’utilisation de cette formulation pour l’encapsulation de chélates de gadolinium ne permet pas d’obtenir des rendements d’encapsulation satisfaisants. La modification du protocole vers une méthodologie Wi/O1/O2ne permet pas d’améliorer l’encapsulation et dénote l’absence d’affinités entre le polymère hydrophobe et les complexes hydrophiles. L’utilisation de nanoparticules composées d’une matrice hydrophile permet d’obtenir des taux de charges nettement supérieurs. Ceci conforte l’hypothèse selon laquelle les interactions entre le complexe et la matrice des nanoparticules jouent un rôle crucial pour l’encapsulation. / The goal of this work was to encapsulate metal complexes into biocompatible nanoparticles for diagnostics. To reach this purpose, a double emulsion-solvent diffusion Wi/O/We technique was optimized, using only biocompatible compounds. It allowed the obtention of PLGA nanoparticles that are compatible with parenteral injections in a reproducible and quantitative way. This formulation was successfully applied to encapsulate model Cu(II)complexes. Optimal formulations showed mean diameters below 200 nm with encapsulation yields in the 20-25 % range. The use of this formulation for gadolinium chelates did not lead to satisfactory encapsulation yields. Thereafter, a Wi/O1/O2 methodology was developed but could not allow to raise the encapsulation efficiencies. This point showed the lack of affinity between the hydrophobic polymer and the hydrophilic chelates. The use of nanoparticles made of an hydrophilic matrix showed a ten-fold increase in the drug loading efficiency. This confirms the hypothesis in which interactions between chelates and nanoparticle matrices play a crucial role for encapsulation.

Two types of (polymer-imaging agent-drug) complexes were prepared and characterized. These included block and graft copolymer complexes with magnetite nanoparticles and manganese ions. Magnetite block ionomer complexes (MBICs) were formed through binding of a portion of the anionic segment of poly(ethylene oxide)-b-poly(acrylic acid) (PEO-b-PAA) block copolymers with the magnetite nanoparticle surfaces. The remainder of the carboxylic acids were utilized to bind with high concentrations of the cationic antibiotic gentamicin (31 wt%). A near zero-order release of gentamicin (pH 7.4 in PBS) that reached ~35 wt% of the initial gentamicin within 10 hours was observed, and this was followed by slower release of another 7 % by 18 hours. These nanoparticles were efficiently taken up by macrophages and appeared to enhance intracellular antimicrobial activities of gentamicin. To increase the complex sizes and NMR T2 relaxivities, amine functional MBICs (MBICs-NH2) were first assembled by adsorbing the polyacrylate block of an aminofunctional poly(ethylene oxide)-b-poly(acrylic acid)) (H2N-PEO-b-PAA) copolymer onto magnetite nanoparticles. Amines at the tips of the H2N-PEO corona were then linked through reaction with a PEO diacrylate oligomer to yield MBIClusters where the metal oxides in the precursor nanoparticles were distinctly separated by the hydrophilic polymer. These MBIClusters with hydrophilic intra-cluster space had transverse relaxivities (r2's) that increased from 190 to 604 s-1 mM Fe-1 measured at 1.4 T and 37°C as their average sizes increased. The clusters were loaded with up to ~38 wt% of the multi-cationic drug gentamicin. MRI scans focused on the livers of mice demonstrated that these MBIClusters are very sensitive contrast agents. These results indicate that these complexes could be potential theranostic agents for dual imaging and drug delivery. Manganese graft ionomer complexes (MaGICs) comprised of Mn ions and a novel polyaminobisphosphonate-g-PEO copolymer were developed for use as T1 weighted MRI positive contrast agents. The graft copolymers were prepared by free radical copolymerization of ammonium bisphosphonate methacrylate monomers with PEO-acrylate macromonomers. The complexes exhibited good colloidal stability without release of free manganese and did not show any in vitro toxicity against mouse hepatocytes. The T1 relaxivities of the MaGICs were 2-10 times higher than that of a commercial manganese based contrast agent MnDPDP. These MaGICs with encapsulated anticancer drugs including doxorubicin, cisplatin and carboplatin have encapsulation efficiencies of 80-100 %. Drug release was sustained and depended on environmental pH, drug structure and drug concentration in the MaGICs. Moreover, these drug-loaded complexes exhibited high anticancer efficacy against MCF-7 breast cancer cells. The prominent MRI relaxivities and high anticancer efficacy suggest that these MaGICs have potential as effective dual imaging and chemotherapeutic agents. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19973号 / 薬科博第64号 / 新制||薬科||7(附属図書館) / 33069 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

Phase-change contrast agents (PCCAs) are an innovative form of imaging agent with practical applications in both the research and clinical settings. PCCAs are derived from gaseous microbubbles, which are able to act as targeted-contrast agents through conjugation of a ligand that is selective for an overexpressed receptor or biomarker in a given disease. Gaseous microbubbles can be condensed to liquid phase nanodroplets, which should be sufficiently small to extravasate into cells and/or tissues given their size and stability. Once liquid nanodroplets have internalized within a given tissue, they can be "activated" back into gaseous microbubbles with ultrasound at clinically used frequencies and energy outputs. This is purposeful as microbubbles provide much greater ultrasound reflectivity than nanodroplets. In this study, PCCAs and/or microbubbles act as a targeting agent in multiple scenarios. The projects in this study include- examination of binding and internalization of targeted PCCAs with different gaseous cores within MDA-MB-231 breast cancer cells, vaporization of liquid phase nanodroplets through application of acoustic energy via focused ultrasound (FUS), and targeting vulnerable plaque in the heart with different types of targeted microbubbles under varying shear-stresses.

Nanoparticles have potential biomedical applications because of their special physical properties. Multi-scale nanoparticles contrast agents are those particles that provide contrast in different scale imaging modalities. Such particles are of great interest for monitoring anti-cancer drug delivery, allowing investigations of whole body drug distribution as well as that at the microscopic level. Magnetic nanoparticles in particular have been used as contrast agents in MRI and optical techniques such as confocal microscopy and two photon fluorescence (TPF) microscopy to achieve multiple scale imaging. However, confocal microscopy has a limited optical penetration in biological tissue. Moreover, both techniques, confocal microscopy and TPF microscopy, require labelling of the biological tissue which alters the particle distribution and complicates the biological system under-investigation.

Dans le cadre du développement des nanotechnologies pour les sciences de la vie et de la santé, les nanovecteurs magnétiques connaissent un essor considérable. Ces structures composites constituées de sphères polymériques encapsulant des nanoparticules magnétiques ou d`un coeur nanoparticulaire magnétique entouré d`une couverture organique présentent une combinaison de propriétés physico-chimiques et magnétiques très performante pour le diagnostic en imageries par exemple, notamment Imagerie par Résonance Magnétique (IRM), ou la thérapie : vectorisation pharmaceutique ciblée, hyperthermie thérapeutique…L`obtention de tels vecteurs avec une taille nanométrique permet l`injection intraveineuse chez les patients et la propagation dans l`organisme, tout en augmentant l`action liée à la surface spécifique. Les présents travaux de doctorat ont porté sur deux familles importantes de nanovecteurs magnétiques : - des nanosphères de polymère biocompatible chargé en composé radioactivable et encapsulant des nanoparticules de magnétite, pour la thérapie tumorale - des agents de contraste pour l`IRM de l`inflammation vasculaire ou cérébrale chez la souris, constituée d`un coeur nanoparticulaire d`oxyde de fer (maghémite ou magnétite) entourée d`une enveloppe organique pour le ciblage de la région visée (ultrasmall superparamagnetic iron oxide – USPIO, en anglais) Pour cerner le comportement de ces nanovecteurs en interaction avec le milieu liquide de suspension, puis avec les régions ciblées dans l`organisme, une approche physique multiéchelle de leurs structure et propriétés a été développée. Les études structurales des nanovecteurs ont été menées à bien grâce à des développements innovants s`appuyant sur les microscopies électroniques à résolution nanométrique. Par l`application du mode Wet-STEM, un nouveau mode en transmission de microscopie électronique à balayage environnementale, l`image en transmission de la structure interne organique/nanoparticule(s) magnétiques(s) a été obtenue et les simulations d`images par méthode de Monte Carlo ont montré qu`une résolution nanométrique pouvait être obtenue. Pour les nanovecteurs en environnement tissulaire, on a utilisé la microscopie électronique en transmission (MET) pour laquelle on a fait varier le degré de coloration dans des préparations de tissus ex vivo inclus en résine ; on a ainsi obtenu les premières images MET en haute résolution (METHR) spatiale d`agents de contraste USPIO cristallisés dans les tissus de l`aorte ou la rate chez la souris athérotique. En combinant ces études structurales avec la mesure des propriétés magnétiques par SQUID, un suivi longitudinal d`agents USPIO injectés chez la souris pour l`IRM de la plaque d`athérome a été menée à bien dans l`aorte et la rate : les résultats ont été interprétés en terme d`agglomération de particules à taille variable en fonction du temps de séjour dans l`organisme et confrontés à un modèle in vitro de dégradation en milieu acide (métabolisme lysosomal). / As applications of nanotechnologies for life and health sciences get booming, magnetic nanovectors undergo a considerable development. Such composite structures made from polymer spheres encapsulating magnetic nanoparticles or from a nanoparticular magnetic core surrounded by an organic coverage exhibit a combination of physical, chemical and magnetic properties very appropriate for diagnostic by imaging such as Magnetic Resonance Imaging (MRI), or for therapy: targeted pharmaceutical vectorization, therapeutic hyperthermia... When such vectors exhibit a nanometric size, intravenous injection and easy spread in the body of the patients are allowed, while effects related to the specific surface area are increased. The present doctoral work was concerned by two important families of magnetic nanovectors: - nanospheres of biocompatible polymer having loaded a radioactivable compound for tumoral therapy and having encapsulated magnetite nanoparticles for diagnostic by MRI: a system for thera-diagnostic is thus obtained.- contrast agents for MRI of vascular or cerebral inflammation, consisting of a nanometric iron oxide (maghemite or magnetite) core i.e. ultrasmall superparamagnetic iron oxide – USPIO - surrounded by an organic coverage for targeting the affected region. These USPIO were used to study inflammation in the atherotic plaque of the aorta in a murine model.Most of the time, such nanovectors are administered to the patients in liquid suspensions by intravenous injection. It is thus crucial to characterize both the collective behaviour and the individual structure of the vectors in liquid suspension. On the other hand their interactions with the targeted regions in the body have to be understood. For this purposes, a multiscale approach of the structure and properties of such nanovectors has been developed, with structural studies carried out through innovative developments based on electron microscopies down to subnanometric resolution and correlated with physical properties. To achieve characterization of nanovectors in liquid media we have developed the application of Wet-STEM, a new mode in transmission of environmental scanning electron microscopy (ESEM), to image the internal structure of the magnetic nanoparticles in liquid suspension and image calculations by Monte Carlo simulations have shown that a nanometric resolution could be theoretically achieved. By the same technique, stability or tendency to flocculation in suspensions can be evidenced with respect to the collective behavior of different nanovectors.In a second step we have investigated the interactions of the nanoparticles with targeted regions. The biodistribution and biotransformation of the USPIO contrast agents in the tissular and cellular environments were investigated at increasing spatial resolution using different techniques. The biodistribution of a MRI contrast agent grafted with a fluorophore, in ex vivo samples from atherotic aorta and spleen were revealed by biphoton microscopy with a resolution of a few hundred nanometers, down to macrophage scale. Then preparation of ex vivo samples for transmission electron microscopy (TEM) was adapted from standard protocols especially with respect to staining after inclusion in resin. This way, the first high resolution HR(TEM) images and electron diffraction patterns of crystallized USPIO contrast agents in the aorta or the spleen of an atheromatous mouse were obtained. Combining such structural studies with measurement (using a SQUID setup) of magnetic properties, a longitudinal follow-up of USPIO nanoparticles injected in mice for MRI of the atherotic plaque has been completed for USPIO particles embedded in the aorta and the spleen: the results were interpreted in terms of agglomeration of the particles with a decreasing size depending on time after injection and found consistent with a model of in vitro degradation in acidic environment proposed to mimick the lysosomal metabolism.

Le scanner spectral à comptage photonique (SPCCT) associé à des agents de contraste spécifiques pourrait détecter certaines inflammations cardiovasculaires à un stade précoce. Les agents de contraste utilisés actuellement en imagerie à rayons X sont des petites molécules qui sont éliminées du système cardiovasculaire en quelques minutes. D’où la nécessité de développer des nanosystèmes présentant des nouvelles caractéristiques intéressantes (ciblage passif/actif, temps de circulation sanguine prolongé, etc.). Dans ce projet de recherche, des nanoparticules (NPs) de polymères iodés ont été produites à l’aide du procédé de nanoprécipitation. Dans un premier temps, les paramètres de procédé et de formulation ont été optimisés pour fournir un agent de contraste répondant à des spécifications physicochimiques précises. Des nanoparticules composées d’une matrice de polymère iodé enveloppée de PEG ont été formulées. Ces suspensions de NPs sont extrêmement stables (jusqu’à 8 mois dans l’eau et dans le sérum humain), monodisperses, avec un diamètre moyen de 150 nm. Une concentration en iode de 100 g(I). mL-1 obtenue après une étape de centrifugation / redispersion a fourni un agent de contraste avec une radiopacité dans la gamme appropriée pour l’imagerie du système cardiovasculaire et l’étude de la biodistribution. Les algorithmes de reconstructions implémentés dans le SPCCT (Décomposition de la matière et reconstruction K-edge), ont permis une quantification précise de l’iode, ainsi qu’une discrimination spécifique du gadolinium et de l’iode dans des phantoms contenant un mélange des deux éléments. La biodistribution a été évaluée après injection des NPs à des rats par voie intraveineuse. Les NPs permettent une visualisation détaillée du système cardio-vasculaire, accompagnée d’une accumulation progressive dans le foie et la rate. Ces deux organes étaient toujours visibles 15 jours après l’injection. Une étude de biodégradation des NPs a été menée sur des cellules de macrophages. Malgré la biodégradabilité potentielle des polymères, les NP intériorisées par les macrophages par phagocytose au cours d’une incubation de 5 heures restent intactes pendant 5 semaines, tandis que certaines cellules sont dégradées. Des NPs ciblant la tropoélastine ont également été synthétisées pour l’imagerie active de la plaque d’athérosclérose au niveau moléculaire. Un test Biacore a montré une affinité des NPs conjuguées avec le peptide de ciblage vis-à-vis de la molécule réceptrice / The Spectral Photon Counting Computed Tomography (SPCCT) technology associated with specific contrast agents could detect some cardiovascular inflammations at an early stage. Currently, contrast agents for CT are small molecules that are eliminated from the cardiovascular system within few minutes. Hence there is a need to develop nanosystems that present new interesting features (passive/active targeting, long blood circulation times, etc.). In this work, iodinated polymer nanoparticles (NPs) were produced using the nanoprecipitation process. First, the process and formulation parameters were optimized to provide a contrast agent that meets definite physicochemical specifications. Highly stable (up to 8 months in water and human serum), monodisperse suspensions of spherical NPs with an average diameter of 150 nm were obtained. The iodinated polymer matrix core is coated by a PEG shell. An iodine concentration of 100 g(I).mL-1 reached after a centrifugation/redispersion step provided radiopacity of the contrast agent in the right range for imaging cardiovascular system and studies of biodistribution. SPCCT material decomposition and K-edge reconstruction allowed accurate quantification of iodine, as well as specific discrimination of gadolinium and iodine in phantoms containing mixtures of both elements. Biodistribution was assessed after intravenous injection of iodinated polymer NPs to rats, revealing a clear visualization of the cardiovascular system, and progressive accumulation in liver and spleen. These organs were still visible up to 15 days post-injection. A biodegradation experiment was carried out on macrophages cell culture. Despite the potential biodegradability of the polymers, NPs internalized by the macrophages after a 5h incubation via the phagocytosis mecanism, remained intact during 5 weeks, while some cells were degraded. Tropoelastin targeting NPs were also developed for active imaging of the atherosclerosis plaque at the molecular level. A Biacore test showed specific affinity of the NPs conjugated with the targeting peptide towards tropoelastin

Optical modulation of the physical properties of materials is important for future development of optical memories and switches, optoelectronics, and smart surfaces. Incorporation of an optically bistable photochromic compound into an electronically bifunctional material is a promising strategy for a development of photoswitchable materials. Photochromic spirooxazine ligands undergo light-induced ring-opening and closure between the closed-spirooxazine (SO) and open-photomerocynanine (PMC) forms. The structural reorganization leads to accompanying changes in electronic structure which can lead to a change in the oxidation/reduction potentials and spin state of a bound metal center. Changes in the ligand field about a metal center in turn can lead to “non-classical” photoinduced magnetic (PIM) effects. The “non-classical” PIM effect is an effect that occurs through ligand-centered processes via the metal center, rather than direct excitation at the metal center. The structural change of the photochromic compounds also results in a change in the frontier orbital energies and donor-acceptor character, which may lead to optically-gated charge-transfer and energy-transfer processes. In this dissertation, the structural factors that govern thermal relaxation of spirooxazines, as optical control units, was investigated toward controlling the photostationary states of this important class of photochromes. The electronic structure of the PMC form of azahomoadamantyl-based spirooxazines was found to control the thermal coloration/decoloration rates of photochromic spirooxazines. A significant charge-separated character of the PMC form was correlated with the slow thermal coloration/decoloration rates in spirooxazines. This concept was then extended to an investigation of the effect of Lewis-acidic metal complexation. Solution study of the charge-separated character of the PMC form via metal complexation of the photochromic spirooxazines supported the correlation between the charge-separated character of the PMC form and the rate of the thermal coloration/decoloration. The studies provide a potential pathway for modulating PMC thermal relaxation rates through optimization of the structure of the spirooxazines and metal complexation. The studies were then extended to an investigation of the photomodulation of charge-transfer processes in cobalt multinuclear clusters by photoisomerization of photochromic spirooxazines. Incorporation of optically bistable phenanthroline-spirooxazine ligands into a magnetically bistable cobalt-dioxolene valence tautomeric cluster resulted in large magnetic moments in the solid and solution states. This study suggests that the redox-isomeric behavior of the cobalt dioxolenes can be coupled to isomerization of the photochromic ligand in the solution state when the π-acceptor ability of the photochromic ligands align with the direction of charge transfer of the cobalt dioxolene components. The potential of these cobalt multinuclear clusters to enhance the relaxivity of water in MRI for biological imaging was investigated. A cobalt tetranuclear cluster was prepared and found to exhibit high magnetic moments in solution at room temperature, and large relaxivities relative to commercially available gadolinium based MRI contrast agents. Lastly, the photomodulation of ionic doping of graphene organic field-effect transistors (OFETs) by photochromic spirooxazines was investigated. The electron donor or acceptor nature of the photochromic isomers modulates the direction and magnitude of ionic doping of graphene, and in turn the gate voltages of graphene OFETs, leading to optical modulation of OFET gate voltages for data processing and memory technologies. / Graduate / 2020-02-08

<p>Ny kunnskap om billeddannelse av hjertet ved magnetisk resonans (MR) fremkommer i sivilingeniør Wibeke Nordhøys doktoravhandling ved Norges teknisk-naturvitenskapelige universitet (NTNU). Denne kunnskapen vil ha stor betydning for hvordan man kan finne fram til noninvasive undersøkelser, dvs. uten fysiske inngrep, av hjertemuskelens levedyktighet hos pasienter med tilstopninger i kransarterier (iskemisk hjertesykdom). </p><p>Mangan som kontrastmiddel og markør for levedyktighet (viabilitet) </p><p>Arbeidet har gitt ny kunnskap om mangan som intracellulær kontrastgiver og om vannutveksling i hjertemuskelen. Manganforbindelser har et stort potensial som kontrastmidler for MR av hjertet. </p><p>I dyreforsøk har Nordhøy vist hvordan det magnetiske sporstoffet mangan tas opp i hjerteceller via fysiologiske ionekanaler, og hvordan det øker kontrasten i MR-bilder ved å «lyse opp hjertecellene innenfra». Hun har også gitt viktige bidrag til forståelsen av hvordan man med MR kan skille mellom vann inne i og utenfor hjertecellene. I sum betyr dette at man har kommet et skritt videre mot at MR av hjertet vil gi gradert informasjon om nettopp hjertecellenes tilstand. Avhandlingen representerer derfor et viktig norsk bidrag til den internasjonale forskning innen molekylær billeddannelse. </p><p>Avhandlingen </p><p>Avhandlingen har tittelen «Manganese and the heart: Intracellular MR relaxation and water exchange across the cardiac cell membrane / Mangan og hjertet: Intracellulær MR relaksasjon og vannutveksling over cellemembranen i hjertet.» Avhandlingen er blitt til innenfor et tverrfaglig samarbeid mellom biofysiker, fysiologer og kjemikere med professor Per Jynge, Institutt for sirkulasjon og bildediagnostikk, NTNU, som hovedveileder og professor Jostein Krane, Kjemisk institutt, NTNU, som medveileder. Arbeidet har vært initiert via Norges forskningsråds strategiske universitetsprogram for medisinsk teknologi (SUP-I) i Trondheim. Det er finansiert av Norges forskningsråd, Det medisinske fakultet (NTNU) og Amersham Health. </p>