Liaison covalente | EPFL Graph Search

Une liaison covalente est une liaison chimique dans laquelle deux atomes se partagent deux électrons (un électron chacun ou deux électrons venant du même atome) d'une de leurs couches externes afin de former un doublet d'électrons liant les deux atomes. C'est une des forces qui produisent l'attraction mutuelle entre atomes. La liaison covalente implique généralement le partage équitable d'une seule paire d'électrons, appelé doublet liant. Chaque atome fournissant un électron, la paire d'électrons est délocalisée entre les deux atomes. Le partage de deux ou trois paires d'électrons s'appelle respectivement « liaison double » et « liaison triple ». Au contraire des liaisons ioniques où les atomes sont liés par attraction coulombienne non-directionnelle, les liaisons covalentes sont fortement directionnelles. En conséquence, les molécules liées par covalence tendent à adopter des formes caractéristiques possédant des angles de liaison spécifiques. Une liaison covalente est une liaison

Preparation and Analysis of Bacteria Surface-Modified with Polymer Nanoparticles

Lisa Patricia Elisabeth Bader

Cell-based drug carriers present an interesting approach to improve the targeted delivery of therapeutic drugs in vivo. Specifically, bacterial cells possess interesting properties such as motility and sensing that allow some strains to selectively target tumor areas. Immobilization of abiotic materials on the cell surface of such bacterial cells can therefore enable the delivery of therapeutic compounds at the desired location. The work presented in this thesis explores the fabrication and characterization of bacteria-nanoparticle biohybrids as potential carriers for drug delivery applications.Chapter 1 presents a review of the scientific literature that covers the different approaches to immobilize abiotic materials on the surface of bacteria. It highlights the advantages of using cells as carriers for the transport of therapeutics. An effective drug delivery agent should present great motility, viability, biodistribution and biocompatibility with human cells. Examples of various chemical approaches to surface-modify bacteria are presented. Chapter 2 systematically explores the immobilization of polystyrene nanoparticles on the surface of Escherichia coli (E. coli) and Salmonella typhimurium (Salmonella) cells. We compared the nanoparticle immobilization using four different conjugations strategies (electrostatic, covalent and ligand-ligand) and characterized the bio-hybrids systems using flow cytometry and confocal microscopy. Two different microscopy techniques were used: the first was based on 3D-reconstruction of z-stacks of single bacterium to investigate position of nanoparticles on the membrane; the second allowed for a large screening of bacteria followed by image processing determining the area of bacteria covered by nanoparticles. We observed that the efficiency of the surface modification is correlated to the initial nanoparticle/cell ratio and that non-covalent strategies allowed for increased number of biohybrids formed and nanoparticle attached. This study provides an overview of the variety of conjugation strategies (and their conjugation efficiencies) available for fabricating nanoparticle-decorated bacteria and presents us an opportunity to better understand and design bacteria-based target-specific drug delivery systems.Chapter 3 presents the characterization of the previously developed bacteria-nanoparticle biohybrids as potential carriers for nanoparticle transport. The viability of the various conjugates was determined by flow cytometry indicating a decrease in cell viability with increasing numbers of nanoparticles immobilized on the membrane. The motility was studied to investigate the impact of nanoparticle attachment on the bacterial velocity as well as the capacity to transport the cargo. Bacteria were able to transport nanoparticles when prepared with low ratios of nanoparticle/cell.

EPFL2022

Identification and characterisation of small-molecule inhibitors targeting STING

Simone Margret Haag

The recognition of pathogen-derived nucleic acids is a critical mechanism by which the innate immune system detects viral infection. Upon activation, nucleic acid sensors initiate the expression of type I interferons and other inflammatory mediators, subsequently leading to potent antiviral immune responses. However, erroneous detection of nucleic acids can have deleterious consequences for the host by inducing the development of certain autoinflammatory disorders. Inappropriate sensing of self- nucleic acids underlies a spectrum of monogenic disease referred to as type interferonopathies, including the Aicardi-Goutières syndrome (AGS). These disorders are often driven by mutations in genes encoding for components of nucleic acid recognition pathways, thereby resulting in the constitutive activation of type I interferon responses. Insight into the molecular pathology of AGS has highlighted promising new targets that may be harnessed for the development of novel therapeutics to counter this disorder. Stimulator of interferon genes (STING) - a central adaptor of the cytosolic DNA sensing pathway - has been strongly implicated in the pathogenesis of distinct inflammatory disorders, including AGS. Therefore, pharmacological inhibition of STING presents a promising approach for the development of potent treatment strategies against these and potentially other STING-mediated, autoinflammatory diseases. Performing a cell-based small-molecule screen, we identified and characterized two series of compounds, which potently and selectively inhibit STING through a mechanism that blocks the activation-induced palmitoylation of STING. The identified small-molecules mediate this inhibition through covalent binding to a transmembrane predicted cysteine residue (Cys91) - a palmitoylation site required for STING activation. Using the inhibitors, we show that the palmitoylation of STING mediates the formation of multimeric complexes at the Golgi and the subsequent recruitment and activation of the direct downstream kinase TBK1. We also provide evidence that representatives of both identified compound classes potently inhibit STING-induced expression of inflammatory cytokines including type I interferon, in human and mouse cells. Furthermore, we also demonstrate that the identified STING inhibitors attenuate autoinflammatory disease features in a mouse model of AGS. Taken together, this work uncovers a small-molecule mediated mechanism to inhibit STING and demonstrates the pharmacological potential of targeting STING for the treatment of type I interferon driven disease such as AGS.

EPFL2019

Chemistry and Analysis of Nanoparticle-Modified Living Cells

Tanja Thomsen

Conventional therapeutics are often limited by their targeting ability, resulting in harmful and potential fatal side-effects for the patients. Recently, new strategies have been developed to improve target specificity of drugs in order to generate more efficient therapeutics. An innovative concept, examined in this thesis, is the use of cells as therapeutic carriers. This concept termed cell-mediated drug delivery, relies on the intrinsic targeting properties of cells to transport the therapeutic drug to the target location. The conjugation of nanoparticles, loaded with drugs or adjuvants, to the surface of cells enables the transport to the desired location and increases the local drug concentration, reducing the required therapeutic leading to further attenuation of side effects. Chapter 1 introduces the concept of cell-mediated drug delivery. It highlights the advantages of using cells decorated with nanoparticles as carriers for therapeutics as opposed to free (polymer) nanoparticles. Examples of various covalent and non-covalent nanoparticle conjugations strategies are discussed and an overview of all approaches, which have been used previously to attach nanoparticles to the cell surface, is presented. The importance of a thorough in vitro characterization of nanoparticle-modified cells is emphasized. The use of common characterization techniques such as flow cytometry, UV-Vis and fluorescence spectroscopy, fluorescence and electron microscopy, and radiolabeling is discussed in detail and examples of qualitative and quantitative in vitro analysis of nanoparticle- modified cells are reviewed. Chapter 2 contains an extensive in vitro analysis of cell-nanoparticle conjugates using fluorescent- and fluorescent label-free methods. The stability of nanoparticles loaded with cargo attached to cells was investigated by encapsulating three different green fluorescent dyes into nanoparticles and tracking the fluorescence signal over 24 h. Two of the dyes revealed a complete loss of fluorescence of a time period of 24 h, whereas a third entrapped dye and a covalently attached dye did not have a decrease in fluorescence over 24 h. Finally, we developed a fluorescent label-free method as an alternative to characterize nanoparticle-decorated cells without the need for any fluorescent label. Chapter 3 systematically explores conjugation chemistries to immobilize two platforms of functional biodegradable polymer nanoparticles (PEG-PLA (poly(ethylene glycol) - poly(lactide)) nanoparticles and carboxylic PLA nanoparticles) on T cells as potential carrier across the blood-brain barrier (BBB). We compared a series of covalent and non-covalent immobilization strategies by flow cytometry (FACS), confocal microscopy, and impact on cell proliferation and cellular viability. We found that T-cell surface coupling of nanoparticles is dependent on the ratio of nanoparticles to cells and that higher ratios led to more particles attached to cells. Furthermore, the most efficient strategy to attach nanoparticles was via ligand-receptor interactions (lectin - sialic acid and biotin - NeutrAvidin). In addition, nanoparticle-decorated T cells were investigated for their ability to cross an in vitro mouse BBB model under static conditions in a two-chamber assay. Decorating the cells with nanoparticles does not affect their ability to bind ICAM-1 (a protein involved in the transmigration process) or to cross the model biological barrier.

EPFL2020