Letteratura scientifica selezionata sul tema "Photo-uncaging"

Abstract Cytokine therapies show great potential to induce cancer immune elimination and have demonstrated therapeutic benefit in a subset of patients. However, controlling the pleotropic effects of these immune signal effectors is a challenge. Here, we present an approach to enable optical control of cytokine activity by caging the activity of these proteins via labeling with wavelength-specific, photo-labile polymers (i.e. photo-caging). We create a system to measure light-induced polymer cleavage (uncaging) through fluorescence dequenching and establish: high wavelength-selectivity, protein activation through tissue mimics, and micrometer-scale uncaging resolution (via photopatterning). We focus on cytokines IL-2, IL-12 and IL-15 and demonstrate ability to reversibly modulate cytokine size and activity. We show photo-caging increases cytokine size by up to 20 fold, resulting in increased serum half-life. Additionally, cytokine activity (via T-cell proliferation) is blunted by 1,000 fold and near fully recovered through light-induced uncaging. Affinity measurements demonstrate photo-caging disrupts the bias of IL-2 towards IL-2Rα expressed on immunosuppressive T-regulatory cells. Further, photo-caging negates the ability of IL-2 to increase antigen specific (OT-1) cell activation, but is restored upon uncaging. In summary, we present the synthesis and therapeutic potential of these optically activated cytokines. In future studies we aim to use cytokine photo-caging as a means to provide new insights into the mechanics of cancer immune elimination and achieve tissue exclusive activation by exploiting spatially and temporally constraining cytokine activation in chip-based organoids and in vivo cancer models.

Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.

Photo-responsive modifications and photo-uncaging concepts are useful for spatiotemporal control of peptides structure and function. While side chain photo-responsive modifications are relatively common, access to photo-responsive modifications of backbone N–H bonds is quite limited. This letter describes a new photocleavage pathway, affording N-formyl amides from vinylogous nitroaryl precursors under physiologically relevant conditions via a formal oxidative C=C cleavage. The N-formyl amide products have unique properties and reactivity, but are difficult or impossible to access by traditional synthetic approaches.

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.

Le cancer est un problème de santé publique majeur : en 2020, il a causé la mort d’environ 10 millions d’individus dans le monde soit l’équivalent d’une mort sur six. Les recherches récentes contre le cancer ont conduit à l’émergence de thérapies innovantes plus ciblées. Ainsi, les dommages collatéraux aux cellules saines et donc les effets secondaires chez les patients traités sont limités, tout en garantissant une efficacité maximale du traitement pour l’éradication de la tumeur. Dans ces travaux de thèse, nous étudions une nouvelle réaction photochimique originale permettant le développement d’une nouvelle prodrogue anti-cancéreuse photoactivable. À partir d’une molécule hélicoïdale de type bis-quinoléine, une photoréaction intramoléculaire de double SNAr* conduit à la formation d’un diazonia étendu, ligand des G-quadruplexes de l’ADN, fluorescent, et capable de générer de l’oxygène singulet cytotoxique. Ainsi, cette réaction photochimique permet le développement d’un traitement anti-cancéreux nouveau à la croisée entre la photothérapie dynamique et le photo-cageage. De plus, la lumière est un stimulus extérieur non-nocif dans une certaine gamme de longueur d’onde (notamment infra-rouge) et qui permet une libération du médicament contrôlé spatio-temporellement ce qui en fait un mode d’action de la prodrogue très ciblé et prometteur. Dans cette thèse, nous avons appréhendé le mécanisme de la photoréaction par étude de l’effet de différents paramètres expérimentaux physico-chimiques. Aussi, nous avons rationalisé l’influence de la nature du groupe partant sur l’efficacité de la double SNAr* et sur le rendement quantique de la photoréaction. Ensuite, nous avons amélioré le design de la prodrogue en vue de son application biologique afin de 1) assurer la biocompatibilité du traitement par déclenchement de la photoréaction dans la fenêtre de transparence biologique 2) améliorer l’internalisation cellulaire de la prodrogue 3) développer une thérapie combinée par association de plusieurs agents anticancéreux et mise en place de stratégies de ciblage
Cancer is a major public health problem: in 2020, it caused the death of approximately 10 million people worldwide, equivalent to one in six deaths. Recent research efforts against cancer have led to the emergence of targeted and innovative therapies. These advancements aim to minimize collateral damage to healthy cells and reduce side effects in treated patients, while ensuring maximum treatment efficacy for tumor eradication. In this thesis, we study a new original photochemical reaction allowing the development of a new photoactivatable anti-cancer prodrug. From a helical molecule of bis-quinoline type, an intramolecular double SNAr* photoreaction leads to the formation of an extended diazonia, a G-quadruplexes ligand which is fluorescent and capable of generating cytotoxic singlet oxygen. Thus, this photochemical reaction facilitates the development of a novel anti-cancer treatment at the intersection of photodynamic therapy and photo-uncaging. Furthermore, light serves as a non-harmful external stimulus within a certain range of wavelengths (particularly infrared), allowing controlled spatiotemporal drug release, rendering the prodrug's mode of action highly targeted and promising. In this thesis, we apprehended the mechanism of the photoreaction by studying the effect of different physico-chemical experimental parameters. Also, we rationalized the influence of the nature of the leaving group on the efficiency of the double SNAr* on the quantum yield of the photoreaction. Then, we improved the design of the prodrug for its biological application in order to 1) ensure treatment biocompatibility by triggering the photoreaction in the biological transparency window, 2) enhance prodrug cellular internalization, and 3) develop a combined therapy by associating multiple anticancer agents and implementing targeting strategies

La photolibération d'agents biologiques cagés par des groupements protecteurs photolabiles (PPGs) a récemment attiré un intérêt croissant en thérapie et physiologie. La combinaison de cet outil avec les avantages de l'absorption à deux photons (2PA) dans le proche IR est cependant un défi. Dans ces travaux, nous présentons deux voies différentes vers de nouveaux décageurs efficaces en 2PA.Nous élaborons d’abord une série de systèmes tandem basés sur le FRET, combinant des antennes quadrupolaires 2PA avec des modules PPGs appropriés. La modification des blocs constitutifs de ces composés nous a permis de moduler les paramètres clés tels que les propriétés photophysiques, l'efficacité de FRET et la cinétique de photolibération. Les systèmes synergiques à base de coumarines, dans lesquels la paire donneur-accepteur est la plus adaptée, ont finalement conduit à des valeurs de δu record pour la libération d’acides carboxyliques.La problématique critique de solubilité en milieu biologique a ensuite été étudiée en incorporant des motifs hydrosolubilisants dans nos systèmes coopératifs. De nouveaux outils hydrophiles et amphiphiles, adaptés respectivement à la délivrance de neurotransmetteurs et la thérapie anticancéreuse, ont ainsi été développés à partir de nos systèmes tandem.Enfin, pour mieux comprendre les relations structure-propriétés dans les PPGs de la famille des coumarines, nous avons synthétisé une série de cages DEAC π-étendues portant différents groupements électroattracteurs, et évalué leur efficacité pour la libération de glycine. Cette étude nous a permis de faire un pas vers la rationalisation du rendement quantique de photolibération chez les PPGs de ce type
Photolabile protecting groups (PPGs) have attracted growing interests in many fields of chemistry and biology. Light-induced release of biological agents, commonly known as “uncaging”, has thus emerged as an interesting process for drug delivery or investigation of biological phenomena. Combining this tool with the intrinsic advantages of two-photon (2P) excitation (2PE) in the NIR is however a challenge. In this work, we use different engineering routes towards new efficient 2P uncagers.First we demonstrate that combining quadrupolar 2P light harvesting antennas with suitable uncaging subunits leads to efficient release of active molecules upon 2PE. In these FRET-based systems, gradual adjustments of the constitutive building blocks allowed us to tune key parameters such as photophysical properties, FRET efficiency, and kinetics of photorelease. In particular, coumarin-based tandem systems, in which absorption and emission of the donor-acceptor pair best match, eventually led to record δu values for uncaging of carboxylic acids.We then assessed the critical water-solubility issue by introducing hydrophilic units onto our cooperative PPGs. New hydrophilic and amphiphilic systems, suited for controlled release of neurotransmitters or anti-cancer agents, were designed from our multi-chromophoric systems.Finally, in our effort towards better understanding of the structure-properties relationships in coumarin PPGs, we synthesized a small library of π-extended DEAC cages bearing strong electron-withdrawing moieties, and assessed their efficacy for 2P uncaging of glycine. With this study, a step was made towards rationalization of the uncaging quantum yield in coumarin cages

Les groupements protecteurs photolabiles (GPPs) sont de petits chromophores organiques capables de libérer des molécules bioactives à la suite d’une irradiation lumineuse. L’utilisation de ces outils attire depuis une quarantaine d’années un intérêt croissant en biologie pour des applications telles que la thérapie ou la physiologie. Bien que performants en excitation à 1-photon, l’élaboration de GPPs performants en excitation à 2- photon (E2P) dans le proche IR reste encore à ce jour un défi de taille. Dans ces travaux, nous choisissons d’adopter deux approches différentes afin d’exalter les propriétés de photolibération des GPPs de la famille des coumarines en E2P.La première résulte d’une modification directe de la structure moléculaire des GPPs afin d’accroitre leur capacité d’absorption à 2-photon (A2P) et donc leur photosensibilité. La modulation des systèmes push-pull, - conjugué et l’introduction de symétrie moléculaire a d’abord permis d’obtenir des GPPs extrêmement performants en E2P. La bibliothèque de composés synthétisés a également permis de faire un pas vers la rationalisation de l’impact des effets électroniques sur le rendement quantique de photolibération. Parmi ceux-ci, un de nos nouveaux GPPs est actuellement sur le point d’être appliqué à des expériences de neurophysiologie.La seconde approche, plus fondamentale, consiste à élaborer de nouveaux systèmes synergiques basés sur des transferts d’énergie de type FRET ou PET entre une antenne aux capacités d’A2P élevées et des GPPs appropriés. Une nouvelle fois, la mise en œuvre de cette stratégie a permis d’atteindre des valeurs de photosensibilité en E2P extrêmement élevée. Par ailleurs, quelques résultats décevants ont néanmoins contribué à mettre en avant les paramètres clés à prendre en considération lors de la conception de tels édifices
Photosensitive protecting groups (PPGs) are small organic chromophores able to release bioactive compounds thanks to light irradiation. Such molecular tools have attracted a growing interest in the field of biology for applications such as therapy or physiology. However, while most PPGs possess satisfying photochemical properties for 1-photon excitation, designing efficient PPGs for 2-photon excitation (2PE) in the NIR region is still very challenging. In this work, we decided to use two different approaches towards new efficient PPGs for two-photon (2P) induced photorelease.First, we decided to investigate the direct modification of the molecular structure of coumarinyl PPGs in order to enhance their 2P absorption (2PA) ability. In this context, the synthesis of PPGs with modified - conjugated and push-pull systems combined with the introduction of molecular symmetry led to a set of very efficient PPGs for 2PE. In addition, a small library of dipolar PPGs bearing electron withdrawing groups allowed us to make a step towards the rationalization of the uncaging quantum yield. One of our new compounds is currently being submitted to neurophysiological experiments to validate our strategy.The second part of this work concerns the conception of energy transfer (FRET or PET) based tandem systems between a strong 2P absorber antennae and appropriate PPG. Again, the photophysical and photochemical studies of our new compounds showed very high 2P photosensitivity. While these last results contributed to validate our strategy, some of the disappointing results obtained also contributed to point out the key parameters required in the elaboration of such architectures

A combined experimental and numerical analysis technique for velocity profiles in liquid microchannel flows is described. The working fluids employed are aqueous solutions with caged fluorescent dyes. A sheet of fluorescent dye is photo-injected by briefly exposing a cross-section of the fluid to ultraviolet light. The transport of the resulting ‘band’ of fluorescent dye is imaged onto a CCD camera using an epi-illumination fluorescent microscope system. The velocity profile is calculated from images acquired and processed after each uncaging event. The analysis technique utilizes several images, to provide velocity data with an increased signal-to-noise ratio. This combined experimental/numerical technique can provide velocity data in the near-wall region as well as in the bulk. Results are shown to compare favorably to analytical solutions in both pressure-driven and electroosmotic flow in circular cross-section capillaries of 205μm and 102μm inner diameters, respectively. Near-wall resolution is verified through application to electroosmotic flows with thin electrical double layers.