Synthesis of cyclodextrin derivatives for practical applications Abstract The first part of this PhD thesis is focused on the synthesis of a series of monosubstituted tetraalkylammonium cyclodextrin (CD) derivatives. The emphasis was placed on the possible applicability of the synthetic process to multigram or even industrial scale. Monotosylation of the native cyclodextrins (-, -, -) on the primary side of the macrocycle afforded the starting materials. Derivatives with one cationic group were prepared by the reaction with aqueous trimethylamine. The reaction of the mono-Ts-CD with neat N,N,N'-trimethylethane-1,2-diamine or N,N,N'-trimethylpropane-1,3-diamine and subsequent methylation led to derivatives with the substituent bearing two cationic groups (PEMEDA- and PEMPDA-β-CD). Analogs bearing a moiety with three tetraalkylammonium sites were synthesized by reaction of mono-Ts-CD with bis(3-aminopropyl)amine with subsequent methylation. 1,3-Dipolar cycloaddition of mono-6- azido--CD with diaminoacetylenes followed by methylation led to analogs with a avariable distance of the charged substituent from the CD core. Majority of the presented reactions are straightforward, relatively high-yielding and the workup does not require chromatographic steps. The second part of the work is dealing with the...

Gene therapy is a potential strategy for treatment of diseases caused by a gene defect. Recent studies are involved particulary in the cure of diseases caused by single gene defect (cystic fibrosis, haemophilia, muscular dystrophy etc.). Our work is part of a project aiming at developing ex vivo non-viral gene delivery systems that could be used for the treatment of ocular and cardiovascular diseases. The gene vectors are biodegradable polymeric carriers based on poly-α-amino acids. These polyplexes should transfect target cells which are supposed to be seeded on polyimide membranes. The biodegradable polymer membrane will be implanted into the retina or used as a coating for cardiovascular prosthesis. As a cover of the implantable membranes we used polymerized methacrylamide-modified gelatin forming hydrogels and mediating a growth support for transfected cells. We focus on material bio- and immunocompatibility/immunoacceptability. The results indicated a very good bio- and immunocompatibility of the gelatin B hydrogel both in vitro and in vivo. The gelatin B hydrogel did not cause erythrocytes lysis, stimulation of proliferation (spontaneous or mitogen-induced) of mouse or human lymphoid cells, neither production of cytokines or NO in vitro. Histological examination following subcutaneous...

Title: Structure and dynamics of electronic defects in liquid water Author: Ondřej Maršálek Institute: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic Supervisor: prof. Mgr. Pavel Jungwirth, DSc. Supervisor's e-mail address: pavel.jungwirth@uochb.cas.cz Abstract: In this thesis we present ab inito molecular dynamics simulations of two different electronic defects in water. Photoionization of liquid water produces a cationic hole, which undergoes ultrafast dynamics and forms the hydrated proton and the hydroxyl radical as its products. We study both the dynamics and spectroscopy of this process. The hydrated electron is a key intermediate in radiation chemistry of aqueous systems. We simulate its equilibrium properties in anionic water clusters as well as the dynamics of vertical electron attachment to cold and warm clusters. The hydrated electron reacts with a hydrated proton to form a hydrogen atom. We examine this reaction at a finite temperature in a larger cluster as well as in more detail in a smaller cluster. Because both of the electronic defects studied here are challenging open-shell species, we put emphasis on benchmarking and testing our computational setup. Six published articles are attached to the thesis. Keywords: density functional theory,...

Les polymères répondant au pH ou à la température deviennent hydrophobes à partir d’un pH ou d’une température critique. Associés à un bloc polymère hydrophile, ils peuvent former des micelles réversibles en solution aqueuse en réponse à un stimulus. Cette thèse décrit principalement la synthèse par polymérisation radicalaire contrôlée de copolymères à blocs triple hydrophiles de type ABC ou ACB, composés d’un bloc très hydrophile (PEO, bloc A), d’un bloc répondant à la température et au pH (PDMAEMA, bloc B) et d’un bloc cationique (PDMAEMAquat, bloc C). Leur caractérisation par chromatographie d’exclusion stérique en phase aqueuse s’est révélée peu concluante ; c’est pourquoi nous les avons analysés par RMN diffusionnelle. D’autre part, l’auto-assemblage en solution aqueuse de ces copolymère triblocs en fonction du pH et de la température a été étudié par RMN 1H et par DLS. Par ailleurs, le bloc C, cationique, forme un complexe hydrophobe avec un polymère chargé négativement (PSS). Les objets résultant de cette complexation entre le dernier bloc du tribloc ABC et le PSS ont été caractérisés par RMN 1H, par DLS, par RMN diffusionnelle et par TEM. Leur comportement en solution aqueuse en fonction du pH et de la température a également été abordé. / PH- or temperature-responsive polymers become hydrophobic from a critical pH or temperature. When they are associated to a hydrophilic block, they may respond to a stimulus by forming reversible micelles in aqueous solution. This thesis mainly deals with the synthesis by controlled radical polymerization of ABC- or ACB-type triple hydrophilic block copolymers, based on a highly hydrophilic block (PEO, A-block), a pH- and temperature-responsive moiety (PDMAEMA, B-block) and a cationic sequence (PDMAEMAquat, C-block). As their characterization by SEC in aqueous phase was not conclusive, they were therefore analyzed by diffusional NMR. In addition, their self-assembly in aqueous solution depending on pH or temperature was studied by 1H NMR and DLS. Furthermore, the cationic C-block form a so-called polyelectrolyte complex with a negatively charged polymer (PSS). Objects resulting from the complexation between the last block of ABC-triblock and PSS were characterized by 1H NMR, DLS, diffusional NMR and TEM. Their behavior in aqueous solution was also investigated depending on pH and temperature.

<p>Pathogenic bacteria are evolving to drug resistant strains at alarming rates. The threat posed by drug resistant bacterial infections emphasize the need to establish new antimicrobial agents. Of immediate concern regarding the dangers of antibiotic resistance is the existence of intracellular bacteria, which find refuge from bactericidal devices by hiding within mammalian cells. Unfortunately, many therapeutics, such as vancomycin, do not possess membrane penetrating abilities to achieve efficacious eradication of bacteria at the subcellular level, allowing infections to persist. In an effort to target pathogens that thrive within mammalian cells, features of cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) were combined to develop a dual action antimicrobial CPP, cationic amphiphilic polyproline helices (CAPHs). CAPHs have proven to be an effective antimicrobial agent to combat an array of both Gram negative and Gram positive bacteria. </p> <p> </p> <p>Herein, to improve CAPHs activity, we have demonstrated how the incorporation of strategic modifications has resulted in increased cell uptake, alternative subcellular locations for CAPHs, and advanced antimicrobial potency. By simultaneously extending the helical length of CAPHs while incorporating different hydrophobic groups in place of the original isobutyl moiety that compose CAPHs we have created a <b>FL-P17-5R </b>series of peptides with five carbon aliphatic motifs: <b>Fl-P17-5B</b>, <b>Fl-P17-5C</b> and <b>Fl-P17-5L. </b>Through these modifications the peptides proved to be 2 to 5-fold more efficient in accumulating in macrophage cells than parent peptide Fl-P14LRR and where able to clear intracellular pathogenic bacteria, such as <i>Listeria</i>, from infected macrophages by 26 to 54%. </p> <p> </p> <p>In addition to making the <b>Fl-P17-5R</b> series of CAPHs to potentiate CAPHs activity, modifications to the cationic moiety of CAPHs were explored. By incorporating a new cationic monomer into the CAPHs sequence, a guanylated amino proline (GAP) residue, we produced <b>Fl-P14GAP</b>, a CAPHs peptide with an organized cationic charge display. This modification resulted in a 5-fold increase in cell uptake and a 2 to 16-fold decrease in minimum inhibitory concentration (MIC) values against strains of enteric and ESKAPE pathogens in comparison to Fl-P14LRR. <b>Fl-P14GAP</b> also executed superior clearance of intracellular pathogenic bacteria that resulted in the complete eradication of a drug resistant strain of <i>A. baumannii</i> from infected macrophage cells. Overall, our efforts with the <b>Fl-P17-5R</b> series of CAPHs and <b>Fl-P14GAP</b> have strengthened the therapeutic potential of CAPHs in the hopes of addressing the need for novel antibiotics with the propensity to eradicate intracellular pathogens.</p>

Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0.

No description available.

No description available.

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.

Le phénomène d’électro chimiluminescence (ECL), également appelé chimiluminescence électrogénérée, consiste en la génération de l’état excité d’un émetteur suite à des réactions de transfert d’électrons se produisant initialement à la surface de l’électrode. L’état excité ainsi produit retourne à l’état fondamental en émettant de la lumière. Les réactions ECL se classent principalement en 2 grandes voies mécanistiques: les réactions d’annihilation et les réactions impliquant un co-réactif sacrificiel. Cette dernière voie a conduit à de très nombreuses applications en chimie analytique. Dans ce manuscrit, j’ai présenté mes travaux de thèse qui ont suivis 3 directions complémentaires depuis l’échelle moléculaire jusqu’à l’échelle macroscopique: la recherche de nouveaux luminophores ECL, l’étude de films d’hydrogels stimulables et le développement de nouvelles applications analytiques de l’ECL.Dans une première partie, j’ai étudié les propriétés ECL de 3 types de luminophores organiques. Ces composés ont montré des caractéristiques électrochimiques et ECL remarquables. L’efficacité ECL de ces luminophores organiques peut être modulée enjouant sur leurs structures respectives. Des luminophores de type spirofluorène ont produit une émission ECL très intense et les nanoparticules organiques correspondantes ont pu être utilisées comme nano-émetteurs ECL. L’étude des propriétés électrochimiques, photochimiques et ECL de luminophores cationiques de type triangulène et hélicène a été réalisée et présentée avec un formalisme montrant un «mur» ECL ou une cartographie ECL complète.Dans une seconde partie, la préparation de films d’hydrogels thermo-stimulables à base de poly(N-isopropylacrylamide) ou pNIPAM incorporant des centres redoxRu(bpy)3 a été réalisée sur des électrodes de carbone vitreux (GCE) et aussi sur des fibres de carbone par polymérisation radicalaire induite électrochimiquement. Les études ECL sur les GCEs modifiées ont montré que le facteur principal gouvernant les propriétés ECL est la distance entre les sites Ru(bpy)3. Le dépôt de tels films de pNIPAM-Ru(bpy)3 par électrochimie bipolaire ouvre de nouvelles possibilités pour le développement de micro-objets stimulables hybrides. Dans une dernière partie, comme la chimie analytique constitue un des plus importants attraits de l’ECL, deux applications analytiques sont présentées en utilisant,d’une part, des co-réactifs de type amine modifié par l’acide phénylboronique, et,d’autre part, des faisceaux de fibres optiques recouverts d’or. La réaction de complexation de saccharides par le groupe phénylboronique modifie les propriétés électrochimiques du co-réactif amine en rendant son oxydation à l’électrode inefficace,ce qui provoque la diminution du signal ECL. En changeant la longueur de l’espaceur de ces co-réactifs qui portent deux groupements phénylboroniques, nous avons pu mesurer sélectivement la concentration de D-glucose et de D-fructose. Mon travail a enfin porté sur le développement d’un objet analytique basé sur un faisceau de fibresoptiques doré qui est adressé sans contact par électrochimie bipolaire. L’ECL ainsi générée du système Ru(bpy)32+/TPrA a permis de réaliser un outil activable à distance permettant une mesure déportée via le faisceau. Ce nouvel objet analytique original devrait permettre d’étendre les mesures ECL à des environnements confinés ou dangereux. / Electrogenerated chemiluminescence (ECL) involves the energetic electron transfer reactions at the electrode with the generation of excited state of emitter, which then relax to the ground state and emit light. These ECL reactions can be divided into two main pathways: the annihilation and sacrificial co-reactant reactions. The latter has found a lot of applications in analytical chemistry. In this thesis, ECL studies towardt hree complementary directions are presented, ranging from the molecular scale tomacroscopic scale : the research of new ECL luminophores, the study of stimuli-responsive hydrogel films, and the development of new ECL assays.Firstly, I have studied three types of organic dyes for ECL investigations. These organic dyes exhibit interesting electrochemical and ECL properties. ECL efficiencies of the organic dyes can be tuned by the modification of the structures. Spirofluorene dyes show strong ECL emission, and thus its fluorescence organic nanoparticles(FONs) prepared in water were used as ECL nano-emitters. We also established an energetic ECL “wall” representation and then move forward creating ECL “map”upon electrochemical, photoluminescence and ECL studies on cationic triangulenes and cationic helicenes dyes, respectively.Secondly, the preparation of thermo-responsive poly(N-isopropylacrylamide)(pNIPAM) hydrogel films covalently incorporating Ru(bpy)3 redox centers were achieved on glassy carbon electrode (GCE) or carbon fiber by electrochemically induced free radical polymerization. ECL studies on the modified GCEs have provided the main factor (the average distance of Ru(bpy)3 sites) that governs the ECL process, leading to deciphering the enhanced ECL in the films. The deposition of the films on carbon fiber by bipolar electrochemistry (BPE) has opened new route to for the development of smart hybrid micro objects. Finally, analytical application is one of the most important features of ECL. We presented two different ECL assays using either the phenylboronic acid modified amine based co-reactants or gold coated optical fiber bundle. The binding of saccharides with boronic acid modified tertiary amines makes the oxidation of amines group inefficient, which decreases ECL signal response. By changing linker length of a bis-boronic acid amine co-reactant, we are able to determine D-glucose and D-fructose selectively. We also studied the ECL generation of Ru(bpy)32+/TPrA systemon the gold coated optical fiber bundle in a wireless manner by BPE, then transmission and remote detection at the opposite end of the same object. This methodmay extend the applicability of ECL assays in the confined or hazardous environments. / 电化学发光（ECL）的发生是由于在电极表面通过电子转移反应生成了发光体的激发态跃迁到基态，并伴随着发光。这些电子转移反应可划分为两种主要的途径：正负自由基湮灭反应和共反应物反应。而后者被广泛应用于分析化学领域。在本论文中，我们在电化学发光领域中进行了广泛的研究，具体有三个研究方向：新型电化学发光光团的研究、响应水凝胶膜的制备以及电化学发光分析的研究。首先，我们选择了三种不同类型的有机荧光分子用于电化学发光的研究。这些有机荧光分子展现出许多电化学和电化学发光特性。其中，螺芴荧光分子展现出了非常强的电化学发光。而且用它制备的荧光有机纳米颗粒（FONs）在水相中也可以产生电化学发光。基于对阳离子型三角烯和阳离子型螺烯的电化学、光谱学以及电化学发光的研究，我们分别建立了鉴别电化学发光“墙”和“图谱”。其次，利用自由基电聚合的方法，我们实现了在玻碳电极和碳纤维表面上制备热刺激-响应的聚异丙基丙烯酰胺（p-NIPAM）共价嫁接三联吡啶钌Ru(bpy)3 荧光分子的水凝胶膜。通过对玻碳电极上水凝胶膜的电化学发光的研究，我们发现了控制水凝胶膜中电化学发光的主要因素，从而揭秘了水凝胶膜中电化学发光增强的成因。而且，利用双电极化学（BPE）的方法，我们将此类水凝胶膜的制备应用于碳纤维上，以发展灵敏杂化微米级器件。最后，鉴于化学分析是电化学发光最重要的特征，我们构建两种不同的电化学发光分析体系：一种是基于硼酸化学修饰的三丙胺共反应物；另一种是利用镀金光导纤维。硼酸对糖类的结合弱化了三丙胺的电化学氧化效率，因此影响电化学发光的强度。通过改变双硼酸修饰共混物之间碳链的长度，我们实现了对葡萄糖和果糖的选择性检测。我们还研究了在镀金光导纤维上三联吡啶钌/三丙胺体系的 电化学发光。此研究是在双电极体系进行的，镀金光导纤维无需外部接线，镀金部位产生的发光透过光纤传输的光纤的远端，再进行检测，因此达到了电化学发光的远程检测。这一方法可应用于狭窄危险环境中的电化学发光分析。