Search

In this study, we explored how chemical reactions of amphiphile compounds can be characterized and followed-up on model interfaces. A custom-made surfactant containing three alkyne sites was first adsorbed and characterized at a water/oil interface. These amphiphiles then underwent interfacial crosslinking by click chemistry upon the addition of a second reactive agent. The monolayer properties and dilatational elasticity, were compared before and after the polymerization. Using bulk phase exchange, the composition of the aqueous bulk phase was finely controlled and washed to specifically measure the interfacial effects of the entities adsorbed and trapped at the interface. In this study, we aim to emphasize an original experimental approach to follow complex phenomena occurring on model interfaces, and also show the potential of this method to characterize multifactorial processes.

Current biomedical applications of nanocarriers are focused on drug delivery, where encapsulated cargo is released in the target tissues under the control of external stimuli. Here, we propose a very different approach, where the active toxic molecules are removed from biological tissues by the nanocarrier. It is based on the drug-sponge concept, where specific molecules are captured by the lipid nanoemulsion (NE) droplets due to dynamic covalent chemistry inside their oil core. To this end, we designed a highly lipophilic amine (LipoAmine) capable of reacting with a free cargo-aldehyde (fluorescent dye and 4-hydroxynonenal toxin) directly inside lipid NEs, yielding a lipophilic imine conjugate well encapsulated in the oil core. The formation of imine bonds was first validated using a push-pull pyrene aldehyde dye, which changes its emission color during the reaction. The conjugate formation was independently confirmed by mass spectrometry. As a result, LipoAmine-loaded NEs spontaneously loaded cargo-aldehydes, yielding formulations stable against leakage at pH 7.4, which can further release the cargo in a low pH range (4-6) in solutions and living cells. Using fluorescence microscopy, we showed that LipoAmine NEs can extract pyrene aldehyde dye from cells as well as from an epithelial tissue (chicken skin). Moreover, successful extraction from cells was also achieved for a highly toxic aliphatic aldehyde 4-hydroxynonenal, which allowed obtaining the proof of concept for detoxification of living cells. Taken together, these results show that the dynamic imine chemistry inside NEs can be used to develop detoxification platforms.

Shape-persistent macrocycles enable superior control on molecular self-assembly, allowing the preparation of well-defined nanostructures with new functions. Here, we report on anionic amphiphilic calixarenes of conic shape and their self-assembly behavior in aqueous media for application in intracellular delivery of peptides. Newly synthesized calixarenes bearing four phosphonate groups and two or four long alkyl chains were found to form micelles of ∼ 10 nm diameter, in contrast to an analogue with short alkyl chains. These amphiphilic calixarenes are able to complex model (oligo-lysine) and biologically relevant (HIV-1 nucleocapsid peptide) cationic peptides into small nanoparticles (20-40 nm). By contrast, a control anionic calixarene with short alkyl chains fails to form small nanoparticles with peptides, highlighting the importance of micellar assembly of amphiphilic calixarenes for peptide complexation. Cellular studies reveal that anionic amphiphilic calixarenes exhibit low cytotoxicity and enable internalization of fluorescently labelled peptides into live cells. These findings suggest anionic amphiphilic macrocycles as promising building blocks for the preparation of peptide delivery vehicles.

The aggregation in a solution of charged dyes such as Rhodamine B (RB) is significantly affected by the type of counterion, which can determine the self-assembled structure that in turn modulates the optical properties. RB aggregation can be boosted by hydrophobic and bulky fluorinated tetraphenylborate counterions, such as F5TPB, with the formation of nanoparticles whose fluorescence quantum yield (FQY) is affected by the degree of fluorination. Here, we developed a classical force field (FF) based on the standard generalized Amber parameters that allows modeling the self-assembling process of RB/F5TPB systems in water, consistent with experimental evidence. Namely, the classical MD simulations employing the re-parametrized FF reproduce the formation of nanoparticles in the RB/F5TPB system, while in the presence of iodide counterions, only RB dimeric species can be formed. Within the large, self-assembled RB/F5TPB aggregates, the occurrence of an H-type RB-RB dimer can be observed, a species that is expected to quench RB fluorescence, in agreement with the experimental data of FQY. The outcome provides atomistic details on the role of the bulky F5TPB counterion as a spacer, with the developed classical FF representing a step towards reliable modeling of dye aggregation in RB-based materials.

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software; a napari plugin for visualization of high-dimensional diffraction-limited polarization camera datasets, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study both alpha-synuclein fibrils and the actin cytoskeleton of mammalian cells. To demonstrate that POLCAM also allows diffraction-limited imaging, we demonstrate POLCAM imaging actin in fibroblast-like cells and the plasma membrane of live human T cells.

International audience; Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

Organelle-specific targeting enables increasing the therapeutic index of drugs and localizing probes for better visualization of cellular processes. Current targeting strategies require conjugation of a molecule of interest with organelle-targeting ligands. Here, we propose a concept of dynamic covalent targeting of organelles where the molecule is conjugated with its ligand directly inside live cells through a dynamic covalent bond. For this purpose, we prepared a series of organelle-targeting ligands with a hydrazide residue for reacting with dyes and drugs bearing a ketone group. We show that dynamic hydrazone bond can be formed between these hydrazide ligands and a ketone-functionalized Nile Red dye (NRK)

We herein present a new concept to produce dual-color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl-coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORM) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC-P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68 nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.

Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ∼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.

To overcome the limited brightness of existing fluorogenic molecular probes for biomolecular targets, we introduce a concept of fluorogenic dendrimer probe, which undergoes polarity-dependent switching due to intramolecular aggregation-caused quenching of its fluorophores. Based on a rational design of dendrimers with four and eight squaraine dyes, we found that octamer bearing dyes through a sufficiently long PEG(8) linker displays >400-fold fluorescence enhancement from water to non-polar dioxane. High extinction coefficient (~2,300,000 M -1 cm -1 ) resulted from eight squaraine dyes and quantum yield (~25%) make this octamer the brightest environment-sensitive fluorogenic molecule reported to date. Its conjugate with cyclic RGD used at low concentration (3 nM) enables integrin-specific fluorescence imaging of cancer cells with high signal-to-background ratio. The developed dendrimer probe is a "golden middle" between molecular probes and nanoparticles, combining small size, turn-on response and high brightness, important for bioimaging.

International audience; Fluorogenic molecular rotors have demonstrated their efficiency to probe viscous environments in various cell compartments. Green emitting BODIPY rotors are non-charged and thus are widely used in bioimaging. However, non-charged rotors operating in the near infrared region and exhibiting high brightness are rare. We herein synthesized five different merocyanines dyes (MCs) from a dihydroxanthene scaffold and studied their spectroscopic properties. Notable differences were observed, which allowed us to establish structure-properties relationships. Among MCs, MC-TB, bearing a thiobarbituric electron acceptor group, displayed high sensitivity to viscosity, excellent photostability, high extinction coefficient (4100 000 M À1 cm À1) and bright emission with sharp peaks in the near infrared region (B700 nm), which are favorable features for bioimaging. In cells, MC-TB stained the most hydrophobic and viscous organelles, namely the lipid droplets. To increase the versatility of this new rotor, a mitochondria targeted version, MC-TB-Mito, was synthesized. The latter showed similar appealing photophysical properties and it was successfully used to report variation and heterogeneity of mitochondrial viscosity using multicolor fluorescence microscopy and fluorescence lifetime imaging (FLIM). Finally, we used MC-TB-Mito to reveal an important increase of mitochondrial viscosity during cell apoptosis.

Nano-emulsions are defined as stable oil droplets sizing below 300 nm. Their singular particularity lies in the loading capabilities of their oily core, much higher than other kinds of carrier. On the other hand, functionalizing the dynamic oil/water interface, to date, has remained a challenge. To ensure the best anchoring of the reactive functions onto the surface of the droplets, we have designed specific amphiphilic polymers (APs) based on poly(maleic anhydride-alt-1-octadecene), stabilizing the nano-emulsions instead of surfactants. Aliphatic C18 chains of the APs are anchored in the droplet core, while the hydrophilic parts of the APs are poly(ethylene glycol) (PEG) chains. In addition, PEG chains are terminated with reactive (i) azide functions in order to prove the concept of the droplet decoration with clickable rhodamine (Rh-DBCO, specifically synthesized for this study), or (ii) biotin functions to verify the potential droplet functionalization with fluorescent streptavidin (streptavidin-AF-488). This study describes AP synthesis, physico-chemical characterization of the functional droplets (electron microscopy), and finally fluorescence labeling and droplet decoration. To conclude, these APs constitute an interesting solution for the stable functionalization of nano-emulsion droplets, paving a new way for the applications of nano-emulsions in targeting drug delivery.

With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

Background Intraoperative localization of endoluminal lesions is can be difficult during laparoscopy. Preoperative endoscopic marking is therefore necessary. Current methods include submucosal tattooing using visible dyes, which in case of transmural injection can impair surgical dissection. Tattooing using indocyanine green (ICG) coupled to intraoperative near-infrared (NIR) laparoscopy has been described. ICG is only visible under NIR-light, therefore, it doesn’t impair the surgical workflow under white light even if there is spillage. However, ICG tattoos have rapid diffusion and short longevity. We propose fluorescent over-the-scope clips (FOSC), using a novel biocompatible fluorescent paint, as durable lesion marking. Methods In six pigs, gastric and colonic endoscopic tattoos using 0.05 mg/mL of ICG and markings using the fluorescent OSC were performed (T0). Simultaneously, NIR laparoscopy was executed. Follow-up laparoscopies were conducted at postoperative day (POD) 4–6 (T1) and POD 11–12 (T2). During laparoscopy, fluorescence intensity was assessed. In one human cadaver, FOSC was used to mark a site on the stomach and on the sigmoid colon, respectively. Intraoperative detection during NIR laparoscopy was assessed. Results Gastric and colonic ICG tattooing and OSC markings were easily visible using NIR laparoscopy on T0. All FOSC were visible at T1 and T2 in both stomach and colon, whereas the ICG tattooing at T1 was only visible in the stomach of 2 animals and in the colon of 3 animals. At T2, tattoos were not visible in any animal. FOSC were still visible in both stomach and colon of the human cadaver at 10 days. Conclusion Endoscopic marking using FOSC can be an efficient and durable alternative to standard methods.

Aim Transanal total mesorectal excision is a promising novel sphincter-saving procedure for low rectal cancer. However, the transanal bottom-up dissection is associated with increased rates of iatrogenic urethral injuries. Near-infrared fluorescence (NIRF) imaging, given its deeper tissue penetration, has been explored in a limited number of studies for enhanced intra-operative urethral visualization. In this study, we explored the feasibility of a novel, ultrabright, biocompatible fluorescent polymer to coat urinary catheters for the purpose of intra-operative urethral visualization. Methods In an ex vivo experiment, using a near-infrared laparoscope, the fluorescent signal of a coated catheter (near-infrared coating of equipment, NICE) was qualitatively and quantitatively compared to the signal of indocyanine green (ICG)/Instillagel® mixtures and ICG-filled catheters at several concentrations. Also, in three male human torsos, using fluorescent urinary catheters, NIRF-guided perineal dissections and a transanal total mesorectal excision were performed. Intra-operative NIRF-based urethral visualization was performed systematically. Results During the qualitative and quantitative fluorescence signal assessment, NICE-coated catheters were clearly superior to the ICG-based solutions. In the cadaveric experiments, enhanced urethral visualization was possible even at early stages of dissection, when the organ was covered by several tissue layers. Conclusions NICE-coated catheters represent a promising potential to allow for NIRF-based intra-operative urethral visualization.

International audience; Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, photostability, lack of a convenient spectral range and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in their push-pull structure raises concerns about their potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push-pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ~100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs. disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400-nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissueslipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.

International audience; Nanoemulsions (NEs) are biocompatible and stealth nanodroplets that can efficiently encapsulate hydrophobic cytoactive drugs in their oily core. NEs were shown to accumulate in tumors by enhanced permeability and retention (EPR) effect and thus display appealing features as nanocarriers to selectively deliver drugs to the tumors. However, to ensure efficient encapsulation with minimal early release, drugs must possess a high degree of lipophilicity. To circumvent this limitation, the latter could be transformed into prodrugs with enhanced hydrophobicity. In return, once delivered in the cell, the prodrug must be efficiently transformed into its active drug form. Herein we chemically and reversibly modified a near infrared Huda dye (HD) into pro-fluorophore (Pro-HD), a non-fluorescent and lipophilic prodrug model that was efficiently loaded in NEs. Thanks to the fluorogenecity of the system (fluorescence enhancement of 35-fold at 723 nm), we demonstrated that Pro-HD did not leak out of NEs, was efficiently delivered into cancer cells and was transformed in cellulo into HD. This proof of concept demonstrates the high potential of lipophilic "pro-fluorophore" approach for visualizing delivery of cargos using NEs as nanocarriers.

International audience; Fluorescent probes are commonly used in studying G protein-coupled receptors in living cells; however their application to the whole animal receptor imaging is still challenging. To address this problem, we report the design and the synthesis of the first near-infrared emitting fluorogenic dimer with environment-sensitive folding. Due to the formation of non-fluorescent H-aggregates in an aqueous medium, the near-infrared fluorogenic dimer displays a strong turn-on response (up to 140-fold) in an apolar environment and exceptional brightness: 56% quantum yield and ≈444 000 M−1 cm−1 extinction coefficient. Grafted on a ligand of the oxytocin receptor, it allows the unprecedented background-free and target-specific imaging of the naturally expressed receptor in living mice.

Nanoprecipitation is a facile and efficient approach to the assembly of loaded polymer nanoparticles (NPs) for applications in bioimaging and targeted drug delivery. Their successful use in clinics requires reproducible and scalable synthesis, for which microfluidics appears as an attractive technique. However, in the case of nanoprecipitation, particle formation depends strongly on mixing. Here, we compare 5 different types of microfluidic mixers with respect to the formation and properties of poly(d-l-lactide--glycolide) (PLGA) and poly(methyl methacrylate) NPs loaded with a fluorescent dye salt: a cross-shaped mixer, a multilamination mixer, a split and recombine mixer, two herringbone mixers, and two impact jet mixers. Size and fluorescence properties of the NPs obtained with these mixers are evaluated. All mixers, except the cross-shaped one, yield NPs at least as small and fluorescent as those obtained manually. Notably in the case of impact jet mixers operated at high flow speeds, the size of the NPs could be strongly reduced from >50 nm down to 70%. These results show the importance of precisely controlling the assembly conditions for loaded polymer NPs. The present work further provides guidance for choosing the optimal microfluidic setup for production of nanomaterials for biomedical applications.

Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.

International audience; Understanding the plasma membrane nanoscale organization and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution that permit for long acquisition times and allow for the quantification of membrane biophysical properties, such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time three-dimensional STED recordings of bleb formation and lipid exchange during membrane fusion as well as by STED-fluorescence correlation spectroscopy experiments for the simultaneous quantification of membrane dynamics and lipid packing that correlate in model and live-cell membranes.

International audience; Dye-loaded polymeric nanoparticles (NPs) are promising bioimaging agents because of their available surface chemistry, high brightness and tunable optical properties. However, high dye loadings can cause the aggregation-caused quenching (ACQ) of the encapsulated fluorophores. Previously, we proposed to mitigate the ACQ inside polymeric NPs by insulating cationic dyes with bulky hydrophobic counterions. In order to implement new functionalities into dye-loaded NPs, here, we extend the concept of bulky counterions to anionic lanthanide-based complexes. We show that by employing Gd-based counterions with octadecyl rhodamine B loaded NPs at 30 wt% vs polymer, the fluorescence quantum yield can be increased 10-fold (to 0.34). Moreover, Gd-anion provides NPs with enhanced contrast in electron microscopy. Combination of a luminescent Eu-based counterion with a far-red to near-infrared cyanine 5 dye (DiD) yields FRET NPs, where the UV-excited Eu-based counterion transfers energy to DiD, generating delayed fluorescence and large stokes shift of ~340 nm. Cellular studies reveal low cytotoxicity of NPs and their capacity to internalize without detectable dye leakage, in contrast to leaky NPs with small counterions. Our findings show that aggregation behavior of cationic dyes in the polymeric NPs can be controlled by bulky lanthanide anions, which will help developing bright luminescent multifunctional nanomaterials.

International audience; Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

Imaging the plasma membrane (PM) by fluorescence techniques using molecular fluorescent probes enable cell segmentation, studying membrane organization and dynamics, formation, and tracking of vesicles. Rational molecular design brings fluorescent PM probes to a new level, providing PM probes with new functions beyond basic PM staining and imaging. We herein review the latest advances in fluorescent PM probes for chemical and biophysical sensing as well as for super-resolution imaging.

Preparation of bright fluorescent materials based on polymers is hampered by a fundamental problem of aggregation-caused quenching (ACQ) of encapsulated dyes. Here, ultrabright fluorescent polymeric nanofibers and coatings are prepared based on a concept of ionic dye insulation with bulky hydrophobic counterions that overcomes the ACQ problem. It is found that bulky hydrophobic counterion perfluorinated tetraphenylborate can boost >100-fold the fluorescence quantum yields of cationic dye octadecyl rhodamine B at high loading (30 wt %) in biocompatible poly(methyl methacrylate) (PMMA). The concept is applicable to both rhodamine and cyanine dyes, which results in bright fluorescent polymeric materials of four different colors spanning from blue to near-infrared. It allows for preparation of electrospun polymeric nanofibers with >50-fold higher dye loading by mass (30 wt %, >20-fold higher molarity for rhodamine dyes) while preserving good fluorescence quantum yields (31%), which implies drastic improvement in their fluorescence brightness. The counterion-based polymeric materials are also validated as coatings of model medical devices, such as stainless steel fiducials and 3D-printed stents of complex geometry. Spin-coated fluorescent polymeric films loaded with a dye paired with bulky counterions exhibit excellent biocompatibility and low toxicity. Moreover, counterion-modified materials show much better stability against dye leakage in the presence of living cells and a serum-containing medium, compared to materials based on the dye with a small inorganic anion. Overall, by pushing the barriers of ACQ, our counterion approach emerges as a powerful tool to develop ultrabright fluorescent polymeric materials ranging from nano- and macroscale.

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution, permitting for long acquisition times, and that allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.