Fluorescence Lifetime Nanoscopy of Liposomal Irinotecan Onivyde: From Manufacturing to Intracellular Processing

: Onivyde was approved by the Food and Drug Administration (FDA) in 2015 for the treatment of solid tumors, including metastatic pancreatic cancer. It is designed to encapsulate irinotecan at high concentration, increase its blood-circulation lifetime, and deliver it to cells where it is enzymatically converted into SN-38, a metabolite with 100- to 1000-fold higher anticancer activity. Despite a rewarding clinical path, little is known about the physical state of encapsulated irinotecan within Onivyde and how this synthetic identity changes throughout the process from manufacturing to intracellular processing. Herein, we exploit irinotecan intrinsic fluorescence and fluorescence lifetime imaging microscopy (FLIM) to selectively probe the supramolecular organization of the drug. FLIM analysis on the manufacturer's formulation reveals the presence of two coexisting physical states within Onivyde liposomes: (i) gelated/precipitated irinotecan and (ii) liposome-membrane-associated irinotecan, the presence of which is not inferable from the manufacturer's indications. FLIM in combination with high-performance liquid chromatography (HPLC) and a membrane-impermeable dynamic quencher of irinotecan reveals rapid (within minutes) and complete chemical dissolution of the gelated/precipitated phase upon Onivyde dilution in standard cell-culturing medium with extensive leakage of the prodrug from liposomes. Indeed, confocal imaging and cell-proliferation assays show that encapsulated and nonencapsulated irinotecan formulations are similar in terms of cell-uptake mechanism and cell-division inhibition. Finally, 2-channel FLIM analysis discriminates the signature of irinotecan from that of its red-shifted SN-38 metabolite, demonstrating the appearance of the latter as a result of Onivyde intracellular processing. The findings presented in this study offer fresh insights into the synthetic identity of Onivyde and its transformation from production to in vitro administration. Moreover, these results serve as another validation of the effectiveness of FLIM analysis in elucidating the supramolecular organization of encapsulated fluorescent drugs. This research underscores the importance of leveraging advanced imaging techniques to deepen our understanding of drug formulations and optimize their performance in delivery applications

New Coumarin Dipicolinate Europium Complexes with a Rich Chemical Speciation and Tunable Luminescence

Europium (III) luminescent chelates possess intrinsic photophysical properties that are extremely useful in a wide range of applications. The lack of examples of coumarin‐based lantha‐ nide complexes is mainly due to poor photo‐sensitization attempts. However, with the appeal of using such a versatile scaffold as antenna, especially in the development of responsive molecular probes, it is worth the effort to research new structural motifs. In this work, we present a series of two new tris coumarin‐dipicolinate europium (III) complexes, specifically tailored to be either a mono or a dual emitter, tuning their properties with a simple chemical modification. We also en‐ countered a rich chemical speciation in solution, studied in detail by means of paramagnetic NMR and emission spectroscopy

New 1,3-Disubstituted Benzo[h]Isoquinoline Cyclen-Based Ligand Platform: Synthesis, Eu3+ Multiphoton Sensitization and Imaging Applications

The development of lanthanide-based luminescent probes with a long emission lifetime has the potential to revolutionize imaging-based diagnostic techniques. By a rational design strategy taking advantage of computational predictions, a novel, water-soluble Eu3+ complex from a cyclen-based ligand bearing 1,3-disubstituted benzo[h]isoquinoline arms was realized. The ligand has been obtained overcoming the lack of reactivity of position 3 of the isoquinoline moiety. Notably, steric hindrance of the heteroaromatic chromophore allowed selective and stoichiometry-controlled insertion of two or three antennas on the cyclen platform without any protection strategy. The complex bears a fourth heptanoic arm for easy conjugation to biomolecules. This new chromophore allowed the sensitization of the metal center either with one or two photons excitation. The suitability as a luminescent bioprobe was validated by imaging BMI1 oncomarker in lung carcinoma cells following an established immunofluorescence approach. The use of a conventional epifluorescence microscope equipped with a linear structured illumination module disclosed a simple and inexpensive way to image confocally Ln-bioprobes by single photon excitation in the 350–400 nm window, where ordinary confocal systems have no excitation sources

Human Microglia Extracellular Vesicles Derived from Different Microglia Cell Lines: Similarities and Differences

ABSTRACT: Microglial cells are a component of the innate immune system in the brain that support cell-to-cell communication via secreted molecules and extracellular vesicles (EVs). EVs can be divided into two major populations: large (LEVs) and small (SEVs) EVs, carrying different mediators, such as proteins, lipids, and miRNAs. The microglia EVs cargo crucially reflects the status of parental cells and can lead to both beneficial and detrimental effects in many physiopathological states. Herein, a workflow for the extraction and characterization of SEVs and LEVs from human C20 and HMC3 microglia cell lines derived, respectively, from adult and embryonic microglia is reported. EVs were gathered from the culture media of the two cell lines by sequential ultracentrifugation steps and their biochemical and biophysical properties were analyzed by Western blot, transmission electron microscopy, and dynamic light scattering. Although the C20and HMC3-derived EVs shared several common features, C20-derived EVs were slightly lower in number and more polydispersed. Interestingly, C20- but not HMC3-SEVs were able to interfere with the proliferation of U87 glioblastoma cells. This correlated with the different relative levels of eight miRNAs involved in neuroinflammation and tumor progression in the C20- and HMC3-derived EVs, which in turn reflected a different basal activation state of the two cell types. Our data fill a gap in the community of microglia EVs, in which the preparations from human cells have been poorly characterized so far. Furthermore, these results shed light on both the differences and similarities of EVs extracted from different human microglia cell models, underlining the need to better characterize the features and biological effects of EVs for therein useful and correct application

Sintesi di nuovi liquidi ionici con nucleo cationico guanidinico

I liquidi ionici sono una classe di composti utilizzati spesso come alternative ecosostenibili ai tradizionali solventi organici. L'enorme variabilità strutturale di questo tipo di composti spinge verso la sintesi di liquidi ionici innovativi, sempre perseguendo gli obbiettivi della "green chemistry". Lo scopo di questo lavoro di tesi cinsiste nella sintesi di liquidi ionici innovativi aventi come catione lo ione guanidinio; per raggiungere tale scopo sono state applicate due strategie sintetiche diverse. Il primo approccio sintetico ha sfruttato la basicità della 1,1,3,3-tetrametilguanidina che per reazione con acidi di Broensted, organici e inorganici, permette di ottenere dei liquidi ionici protici, aventi nucleo guanidinico. Nel secondo approccio sono stati sintetizzati dei sali alogenuri di esaalchilguanidinio partendo da delle tetraalchiluree (la 1,1,3,3,-tetrametilurea e la 1,3-dimetilimidazolidinone); inoltre attraverso reazioni di, metatesi è stato effettuato lo scambio dell'anione di partenza al fine di valutare l'influenza del anione sulle caratteristiche fisiche di questi liquidi ionici. Infine tutti i liquidi ionici sintetizzati sono stati caratterizzati tramite analisi NMR e FT-IR. Inoltre di alcuni campioni sono state effettuate analisi termo-gravimetriche al fine di investigare la stabilità termica

Pioglitazone-Loaded PLGA Nanoparticles: Towards the Most Reliable Synthesis Method

Recent findings have proved the benefits of Pioglitazone (PGZ) against atherosclerosis and type 2 diabetes. Since the systematic and controllable release of this drug is of significant importance, encapsulation of this drug in nanoparticles (NPs) can minimize uncontrolled issues. In this context, drug delivery approaches based on several poly(lactic-co-glycolic acid) (PLGA) nanoparticles have been rising in popularity due to their promising capabilities. However, a fully reliable and reproducible synthetic methodology is still lacking. In this work, we present a rational optimization of the most critical formulation parameters for the production of PGZ-loaded PLGA NPs by the single emulsification-solvent evaporation or nanoprecipitation methods. We examined the influence of several variables (e.g., component concentrations, phases ratio, injection flux rate) on the synthesis of the PGZ-NPs. In addition, a comparison of these synthetic methodologies in terms of nanoparticle size, polydispersity index (PDI), zeta potential (ζp), drug loading (DL%), entrapment efficiency (EE%), and stability is offered. According to the higher entrapment efficiency content, enhanced storage time and suitable particle size, the nanoprecipitation approach appears to be the simplest, most rapid and most reliable synthetic pathway for these drug nanocarriers, and we demonstrated a very slow drug release in PBS for the best formulation obtained by this synthesis

Quantitative determination of fluorescence labeling implemented in cell cultures

Abstract Background Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative. Results We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, and reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4′-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling. Conclusions The developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions

Coumarin-based fluorescent biosensor with large linear range for ratiometric measurement of intracellular pH

Intracellular pH is a critical parameter involved in cell machinery, and its dysregulation can either cause or signal pathologic states. Currently described fluorescent pH probes are based on single acid-base equilibria, and for this reason are intrinsically unable to capture the wide range of cell pH, usually spanning over more than four units. Here we describe a fluorescent pH biosensor based on a conjugated coumarin-triazine scaffold that is excitable in the visible range, shows pseudo-linear ratiometric response over more than 6 pH units with a single fluorogenic unit, and allows imaging of the whole endo-lysosomal pH landscape of living cells with a single acquisition. The probe can discriminate, on the basis of intracellular acidity, between physiologic and tumor cells, being potentially suitable in perspective as diagnostic tool

Investigation of a family of structurally-related guanidinium ionic liquids through XPS and thermal analysis

A family of structurally-related guanidinium bistriflimide ionic liquids has been prepared and characterized. TGA analyses showed a high thermal stability for all the proposed ionic liquids while DSC and XPS analyses divided them into distinct subsets depending on whether one or more constraining cycles were present. The results obtained highlighted the influence of the cation structure on some of the physico-chemical properties and thus the possibility to tune them by selecting proper substituents. The solvatochromic parameters of a selected guanidinium IL have also been studied. (C) 2018 Elsevier B.V. All rights reserved

Uranium-free X solution: a new generation contrast agent for biological samples ultrastructure

Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power. For Transmission Electron Microscopy analysis, biological samples are generally embedded in resins, which allow thin sectioning of the specimen. Embedding resins are also composed by light atoms, thus the contrast difference between the biological sample and the surrounding resin is minimal. Due to that reason in the last decades, several staining solutions and approaches, performed with heavy metal salts, have been developed with the purpose of enhancing both the intrinsic sample contrast and the differences between the sample and resin. The best staining was achieved with the uranyl acetate (UA) solution, which has been the election method for the study of morphology in biological samples. More recently several alternatives for UA have been proposed to get rid of its radiogenic issues, but to date none of these solutions has achieved efficiencies comparable to UA. In this work, we propose a different staining solution (X Solution or X SOL), characterized by lanthanide polyoxometalates (LnPOMs) as heavy atoms source, which could be used alternatively to UA in negative staining (NS), in en bloc staining, and post sectioning staining (PSS) of biological samples. Furthermore, we show an extensive chemical characterization of the LnPOM species present in the solution and the detailed work for its final formulation, which brought remarkable results, and even better performances than UA