Publications

Background/Objectives: Local delivery of gene therapy products through the airways shows great promise for the treatment of a number of serious lung diseases, but its effectiveness is hampered by the mucus layer protecting the lung epithelium in the trachea and bronchi. Methods: To overcome this barrier, we engineered carbon dots (CDs) with mucus penetrating properties. Results: The CDs were synthesized by solvothermal treatment of citric acid and branched polyethyleneimine, and functionalized with maleamic acid groups to create cationic mucoinert nanoparticles with tunable charge. We characterized their interactions with a mucus model through turbidity and transport measurements, and assessed their impact on the viscoelastic properties of the biopolymer. We then demonstrated that the carriers are effective at delivering pDNA to a variety of cell models in vitro. In particular, mucus-producing Calu-3 cells cultured at the air–liquid interface (ALI) were used as a discriminating model to evaluate intracellular delivery of the genetic cargo through a thick layer of mucus at the cell surface. Conclusions: The functionalization of CDs with maleamic acid groups resulted in a 1000- to 10,000-fold increase in transfection efficiency in the mucus-producing model, offering new opportunities for lung gene therapy.

Cationic polyamine-based carbon dots (CDs) are increasingly being explored for biomedical applications. These ultrasmall (&lt;10 nm) fluorescent nanoparticles, synthesized from organic precursors and functionalized with polyamines, possess a strong positive surface charge that enables efficient complexation and delivery of nucleic acids, making them promising candidates for gene therapy. However, the mechanisms by which the immune system, particularly macrophages, recognizes and responds to these nanomaterials remain poorly understood. In this study, we investigated the role of surface receptors in the uptake and biological effects of cationic polyamine-based CDs in macrophages. Our data showed that Fc receptors and the Toll-like receptor 4 (TLR4) were minimally involved in CD internalization and associated cellular responses in contrast to scavenger receptors (SRs). Indeed, SR inhibition reduced CD-induced cell viability loss, LDH release, and secretion of the pro-inflammatory cytokine IL-1β. Among SRs, SR-A1 was identified as a key receptor mediating CD recognition and toxicity, likely through activation of the MERTK signaling pathway. Importantly, these mechanisms occurred in the absence of serum, indicating that protein corona formation is not required for CD interaction with macrophage surface receptors. Overall, our findings highlight the prominent role of SRs, particularly SR-A1, as receptors recognizing cationic polyamine-based CDs on the surface of macrophages, and provide new insights into the cellular mechanisms underlying the immunotoxicity of these carbon-based nanomaterials.</p></div>

Abstract In human cells, large multi-protein transcription co-activators, such as chromatin remodelers or histone acetyltransferases, play critical roles in gene-expression regulation and are often implicated in disease. However, interrogating their structures or analyzing their properties and interactions in different organs or in medically relevant cell-types is hindered by the difficulty in purifying them. We overcome this difficulty by applying an affinity-ligand composed of a small molecule that specifically recognizes a particular domain in a given co-activator multiprotein complex. This molecule is coupled to a desthiobiotin moiety, which allows binding to streptavidin beads and can be eluted using biotin. To demonstrate the universal utility of this idea to practically any co-activator complex from any cell source we synthesized a compound conjugating desthiobiotin to GSK4027, a molecule that targets the bromodomain in the GCN5/PCAF catalytic subunit of SAGA and ATAC acetyltransferase complexes. Employing this heterobifunctional affinity ligand and a novel purification scheme adapted to low-abundance complexes, we isolated the 1.6 MDa SAGA complex from two cancer cell lines to high degree of purity and activity. We then solved the structure of the isolated 20-subunit SAGA complex to high-resolution (2.3-3 Å) by cryo-EM, elucidating for the first time the molecular details of how its enigmatic splicing module anchors into SAGA. Analyzing these interactions raises the possibility that SAGA serves to relay this module to the splicing machinery. Our approach will be instrumental for characterizing many other multi-protein complexes from medically important sources.

Emerging evidence suggests that the function and position of organelles are pivotal for tumor cell dissemination. Among them, lysosomes stand out as they integrate metabolic sensing with gene regulation and secretion of proteases. Yet, how their function is linked to their position and how this controls metastasis remains elusive. Here, we analyze lysosome subcellular distribution in patient-derived melanoma cells and patient biopsies and show that lysosome spreading scales with melanoma aggressiveness. Peripheral lysosomes promote matrix degradation and cell invasion which is directly linked to the lysosomal and cell transcriptional programs. Using chemo-genetical control of lysosome positioning, we demonstrate that perinuclear clustering impairs lysosome secretion, matrix degradation and invasion. Impairing lysosome spreading significantly reduces invasive outgrowth in two in vivo models, mouse and zebrafish. Our study provides a direct demonstration that lysosome positioning controls cell invasion, illustrating the importance of organelle adaptation in carcinogenesis and suggesting its potential utility for diagnosis of metastatic melanoma.</p><p>Metastases are responsible for the majority of cancer-related deaths 1 . Melanoma shows strong negative correlation between cancer stage and 5-year patient survival, making it an ideal model to study phenotypic changes leading to cancer cell invasion, adaptation and survival. Melanoma progression consists of multiple sequential events and its early detection is key for patient survival. First, melanocytes are transformed and grow in the epidermis during radial growth phase (RGP), forming a lesion with low potential to develop metastasis. Changes in their transcription program lead to expression of matrixdegrading enzymes and to invasion through the dermis during vertical growth phase (VGP) followed by cancer dissemination through vascular and lymphatic routes, progressing into metastatic stages 2 .

ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP , an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively biotinylates endogenous P2X7 in BV2 cells, a murine microglia model, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1β release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level.

New concepts to treat eye diseases have emerged that elegantly combine unnatural light exposure with chemical biology approaches to achieve superior cellular specificity and, as a result, improvement of visual function. Historically, light exposure without further molecular eye treatment has offered limited success including photocoagulation to halt pathological blood vessel growth or low light exposure to stimulate retinal cell viability. To add cellular specificity to such treatments, researchers have introduced various biological or chemical light-sensing molecules and combined those with light exposure. (Pre-) clinical trials describe the use of optogenetics and channelrhodpsins, i.e. light-sensitive ion channels, in patient vision restoration. In the chemical arena, pharmacological agents, rendered light-sensitive by reversible modification with photosensitive protecting compounds (“caging”), have been applied to eyes of living mice to photo-release specific cellular activities. Among these were successful proof-of-principle experiments that were conducted to establish photo-sensitive gene therapies in the eye. For light-mediated treatment in combination with chemical biology, we wish to describe here the current frontiers of research in vision restoration with an eye on differences between biological and chemical light-sensing molecules, patient requirements, and future outlooks.

We synthesized new para-dialkylaminonitrobiphenyl (ANBP) derivatives, s-ANBP and t-ANBP, functionalized with dimethyltrityl and phosphoramidite groups for incorporation into DNA backbones as photocleavable linkers via solid-phase synthesis. Both derivatives exhibited excellent chemical stability under diverse conditions, including acidic, alkaline, and high-salt environments and elevated temperatures. Their incorporation into DNA influenced duplex stability and antisense oligonucleotide (ASO) dissociation efficiency, depending on the number of ANBP units and adjacent nucleotide deletions. The s-/t-ANBPconjugated DNA showed efficient one-photon photolysis at 415 nm and enhanced two-photon absorption for extended π-system in t-ANBP, with δ_uΦ_u values of 1.6 GM (740 nm) and 2.7 GM (800 nm). ANBP-conjugated DNA was used to construct a 3D DNA nanocage capable of light-triggered ASO 4625 release, validated by an in vitro RNA digestion assay, confirming antisense functionality. This platform demonstrates precise, light-mediated therapeutic delivery and offers potential for broader applications in drug delivery and clinical use.

Although lung gene therapy holds promise for treating various life-threatening lung diseases, its efficacy is hindered by the mucus layer covering the airways, whose role is to protect the lung epithelium from airborne threats. For efficient gene delivery to the epithelial cells, it is necessary to ensure rapid passage of the transfection particles through the mucus layer before they are eliminated by mucociliary clearance. We developed mucus-penetrating gene carriers using carbon dots (CDs) synthesized from citric acid and bPEI600. Various strategies were investigated to convert these CDs into muco-inert nanoparticles, including PEGylation and decoration with zwitterionic or mucolytic species. After thorough characterization, we assessed their interactions with a mucus model through turbidimetry and transport measurements, as well as their effects on mucus rheology. The efficacy of the carriers to deliver DNA to various cell models was established. Particularly, Calu-3 cells, cultured at the air-liquid interface to obtain abundant mucus production, were used as a discriminating model to evaluate the potency of CDs to deliver their DNA cargo through mucus. While zwitterion-coated CDs failed to induce significant transgene expression, those with PEG decorations yielded moderate results, and CDs designed as thiol reservoirs for local mucolytic action achieved high transfection rates.

The retina is prone to developing pathological neovascularization, a leading cause of blindness in humans. Because excess neovascularization does not affect the entire retina, global inhibition treatment of angiogenesis critically interferes with healthy, unaffected retinal tissue. We therefore established an in vivo photoactivated gene expression paradigm which would allow light-mediated targeting of antiangiogenic genetic treatment only to affected retinal regions. We synthesized a “caged” (i.e., reversibly inhibited) photosensitive 4-hydroxytamoxifen analog. Molecular docking analyses validated its reduced transcriptional activity. Caged 4-hydroxytamoxifen was intravitreally injected into mice harboring the inducible Cre/lox system, with CreERT2 being expressed via the Tie2 promoter in the neurovasculature. Subsequent in vivo irradiation of eyes significantly induced retinal expression of a Cre-dependent transgene in retinal blood vessels. Using GFAP-CreERT2 mice, successful photoactivation was also achieved in eyes and also in ex vivo brain slices for validation of the approach. This highlights the possibility of light-mediated gene therapies specific for the retina, a key first step in personalized medicine.

The emerging field of photopharmacology is a promising chemobiological methodology for optical control of drug activities that could ultimately solve the off-target toxicity outside the disease location of many drugs for the treatment of a given pathology. The use of photolytic reactions looks very attractive for a light-activated drug release but requires to develop photolytic reactions sensitive to red or near-infrared light excitation for better tissue penetration. This review will present the concepts of triplet-triplet annihilation upconversion-based photolysis and their recent in vivo applications for light-induced drug delivery using photoactivatable nanoparticles.

The physiological problem of chronic inflammation and its associated pathologies attract ongoing attention with regard to methods for their control. Current systemic pharmacological treatments present problematic side effects. Thus, the possibility of new anti-inflammatory compounds with differing mechanisms of action or biophysical properties is enticing. Cationic polymers, with their ability to act as carriers for other molecules or to form bio-compatible materials, present one such possibility. Although not well described, several polycations such as chitosan and polyarginine, have displayed anti-inflammatory properties. The present work shows the ubiquitous laboratory transfection reagent, polyethylenimine (PEI) and more specifically low molecular weight branched PEI (B-PEI) as also possessing such properties. Using a RAW264.7 murine cell line macrophage as an inflammation model, it is found the B-PEI 700 Da as being capable of reducing the production of several pro-inflammatory molecules induced by the endotoxin lipopolysaccharide. Although further studies are required for elucidation of its mechanisms, the revelation that such a common lab reagent may present these effects has wide-ranging implications, as well as an abundance of possibilities.

Increasingly, retinal pathologies are being treated with virus-mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e., ‘caging.’ Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP-CreERT2 mice with a Cre-dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light-mediated gene therapy for the eyes.

Liposome-based nanoparticles able to release, via a photolytic reaction, a payload anchored at the surface of the phospholipid bilayer were prepared. The liposome formulation strategy uses an original drug-conjugated blue light-sensitive photoactivatable coumarinyl linker. This is based on an efficient blue light-sensitive photolabile protecting group modified by a lipid anchor, which enables its incorporation into liposomes, leading to blue to green light-sensitive nanoparticles. In addition, the formulated liposomes were doped with triplet–triplet annihilation upconverting organic chromophores (red to blue light) in order to prepare red light sensitive liposomes able to release a payload, by upconversion-assisted photolysis. Those light-activatable liposomes were used to demonstrate that direct blue or green light photolysis or red light TTA-UC-assisted drug photolysis can effectively photorelease a drug payload (Melphalan) and kill tumor cells in vitro after photoactivation.

This work describes the reactivity and properties of fluorinated derivatives (F-PD and F-PDO) of plasmodione (PD) and its metabolite, the plasmodione oxide (PDO). Introduction of a fluorine atom on the 2-methyl group markedly alters the redox properties of the 1,4-naphthoquinone electrophore, making the compound highly oxidizing and particularly photoreactive. A fruitful set of analytical methods (electrochemistry, absorption and emission spectrophotometry, and HRMS-ESI) have been used to highlight the products resulting from UV photoirradiation in the absence or presence of selected nucleophiles. With F-PDO and in the absence of nucleophile, photoreduction generates a highly reactive ortho-quinone methide (o-QM) capable of leading to the formation of a homodimer. In the presence of thiol nucleophiles such as β-mercaptoethanol, which was used as a model, o-QMs are continuously regenerated in sequential photoredox reactions generating mono-or disulfanylation products as well as various unreported sulfanyl products. Besides, these photoreduced adducts derived from F-PDO are characterized by a bright yellowish emission due to an excited-state intramolecular proton transfer (ESIPT) process between the dihydronapthoquinone and benzoyl units. In order to evidence the possibility of an intramolecular coupling of the o-QM intermediate, a synthetic route to the corresponding anthrones is described. Tautomerization of the targeted anthrones occurs and affords highly fluorescent stable hydroxyl-anthraquinones. Although probable to explain the intense visible fluorescence emission also observed in tobacco BY-2 cells used as a cellular model, these coupling products have never been observed during the photochemical reactions performed in this study. Our data suggest that the observed ESIPT-induced fluorescence most likely corresponds to the generation of alkylated products through reduction species, as demonstrated with the β-mercaptoethanol model. In conclusion, F-PDO thus acts as a novel (pro)-fluorescent probe for monitoring redox processes and protein alkylation in living cells.

Abstract Cell invasion is a highly complex process that requires the coordination of cell migration and degradation of the extracellular matrix. In melanoma cells, as in many highly invasive cancer cell types these processes are driven by the regulated formation of adhesives structures such as focal adhesions and invasive structures like invadopodia. Structurally, focal adhesion and invadopodia are quite distinct, yet they share many protein constituents. However, quantitative understanding of the interaction of invadopodia with focal adhesion is lacking, and how invadopodia turn-over is associated with invasion-migration transition cycles remains unknown. In this study, we investigated the role of Pyk2, cortactin and Tks5 in invadopodia turnover and their relation with focal adhesions. We found that active Pyk2 and cortactin are localised at both focal adhesions and invadopodia. At invadopodia, localisation of active Pyk2 is correlated with ECM degradation. During invadopodia disassembly, Pyk2 and cortactin but not Tks5 are often relocated at nearby nascent adhesions. We also show that during ECM degradation, cell migration is reduced which is likely related to the sharing of common molecules within the two structures. Finally, we found that the dual FAK/Pyk2 inhibitor PF-431396 inhibits both focal adhesion and invadopodia activities thereby reducing both migration and ECM degradation.

Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamages, while allowing to image about 10 times deeper than with a confocal. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. These probes react with bicyclo[6.1.0]no-4-yn-9yl)methanol (BCN) with good to impressive kinetic rate constant (up to 1.1x103 M-1.s-1) and emit in the red window with moderate to high turn-on. TDDFT allowed to rationalize both the kinetic and fluorogenic performance of the different probes. The best candidate displays a 13.8-fold turn-on measured by quantifying fluorescence intensities in live cells under one-photon excitation, whereas a value of 3 is sufficient for high contrast live-cell imaging. In addition, live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe since a 8.0-fold turn-on was measured under two-photon excitation. Finally, the high two-photon brightness of the clicked adduct (>300 GM) allows the use of a weak laser power compatible with in vivo imaging.

Carbon dots (CDs) are nanoparticles (NPs) with potential applications in the biomedical field. When in contact with biological fluids, most NPs are covered by a protein corona. As well, upon cell entry, most NP are sequestered in the lysosome. However, the interplay between the lysosome, the protein corona and the biological effects of NPs is still poorly understood. In this context, we investigated the role of the lysosome in the toxicological responses evoked by four cationic CDs exhibiting protonatable or non-protonatable amine groups at their surface, and the associated changes in the CD protein corona. The four CDs accumulated in the lysosome and led to lysosomal swelling, loss lysosome integrity, cathepsin B activation, NLRP3 inflammasome activation, and cell death by pyroptosis in a human macrophage model, but with a stronger effect for CDs with titratable amino groups. The protein corona formed around CDs in contact with serum partially dissociated under lysosomal conditions with subsequent protein rearrangement, as assessed by quantitative proteomic analysis. The residual protein corona still contained binding proteins, catalytic proteins, and proteins involved in the proteasome, glycolysis, or PI3k-Akt KEGG pathways, but with again a more pronounced effect for CDs with titratable amino groups. These results demonstrate an interplay between lysosome, protein corona and biological effects of cationic NPs in link with the titratability of NP surface charges.

Agonists of the β2 adrenergic receptor (ADRB2) are an important class of medications used for the treatment of respiratory diseases. They can be classified as short acting (SABA) or long acting (LABA), with each class playing a different role in patient management. In this work we explored both ligand-based and structure-based high-throughput approaches to classify β2-agonists based on their duration of action. A completely in-silico prediction pipeline using an AlphaFold generated structure was used for structure-based modelling. Our analysis identified the ligands’ 3D structure and lipophilicity as the most relevant features for the prediction of the duration of action. Interaction-based methods were also able to select ligands with the desired duration of action, incorporating the bias directly in the structure-based drug discovery pipeline without the need for further processing.

In this study, we synthesized two phosphoramidites based on (2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene (BNSF) and (4,4’-bis-{8-[4-nitro-3-(2-propyl)-styryl]}-3,3’-di-methoxybiphenyl (BNSMB) structures as visible light-cleavable linkers for oligonucleotide conjugation. In addition to commercial ultraviolet (UV) photocleavable (PC) linker, the BNSMB linker was further applied as a building component to construct photoregulated DNA devices as duplex structures which are functionalized with fluorophores and quenchers. Selective cleavage of PC and BNSMB is achieved in response to ultraviolet (UV) and visible light irradiations as two inputs respectively. This leads to controllable dissociation of pieces of DNA fragments which is followed by changes of fluorescence emission as signal outputs in of system. By tuning the number and position of the photocleavable molecules, fluorophores and quenchers, various DNA devices were developed in which they mimic functions of Boolean logic gates and achieve logic operations in AND, OR, NOR and NAND gates in response to two different wavelengths of light inputs. By sequence design, the photolysis products can be precisely programmed in DNA devices and triggered release in a selective or sequential manner. Thus, this photoregulated DNA device shows potentials as a wavelength dependent drug delivery system for selective control over the release of multiple individual therapeutic oligonucleotide-based drugs. We believe that our work not only enriches the library of photocleavable phosphoramidite available for bio-conjugation, but also pave the way of developing spatiotemporal-controlled, orthogonal regulated DNA-based logic circuits for a range of applications in materials science, polymer, chemistry and biology.

In this first quantitative proteomic analysis of the protein corona formed on Carbon dots (CDs), we showed that protein corona identity is influenced by CD surface charge properties, which in turn impacts CD uptake and viability loss in macrophages.

Photolytic reactions allow the optical control of the liberation of biological effectors by photolabile protecting groups. The development of versatile technologies enabling the use of deep-red or NIR light excitation still represents a challenging issue, in particular for light-induced drug release (eg; light induced prodrug activation). Here, we present light-sensitive biocompatible lipid nanocapsules able to liberate an antitumoral drug through photolysis. We demonstrate that original photon upconverting nanoparticles (LNC-UCs) chemically conjugated to a coumarin-based photocleavable linker can quantitatively and efficiently release a drug by upconversion luminescence-assisted photolysis using a deep-red excitation wavelength. In addition, we were also able to demonstrate that such nanoparticles are stable in the dark, without any drug leakage in the absence of light. These findings open new avenues to specifically liberate diverse drugs using deep- red or NIR excitations for future therapeutic applications in nanomedicine.

Carbon nanomaterials, including carbon dots (CDs), form a growing family of engineered nanoparticles (NPs) with widespread applications. As the rapid expansion of nanotechnologies raises safety concerns, interaction of NPs with the immune system is receiving a lot of attention. Recent studies have reported that engineered NPs may induce macrophage death by pyroptosis. Therefore, this study investigated whether cationic CDs induce pyroptosis in human macrophages and assessed the role of inflammasome and lysosome in this process. Cationic CDs were synthetized by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine. The NPs evoked a dose-dependent viability loss in THP-1-derived macrophages. A cell leakage, an increase in IL-1β secretion and an activation of caspase-1 were also observed in response to the NPs. Inhibition of caspase-1 decreased CDinduced cell leakage and IL-1β secretion, while restoring cell viability. Besides, CDs triggered swelling and loss of integrity of lysosome, and inhibition of the lysosomal enzyme cathepsin B decreased CD-induced IL-1β secretion. Thus, our data provide evidence that cationic CDs induce inflammasome-dependent pyroptosis in macrophages via lysosomal dysfunction.

Red to NIR absorption and emission wavelengths are key requirements for intravital bioimaging. One of the way to reach such excitation wavelengths is to use two-photon excitation. Unfortunately, there is still a lack of two-photon excitable fluorophores that are both efficient and biocompatible. Thus, we design a series of biocompatible quadrupolar dyes in order to study their ability to be used for live-cell imaging, and in particular for two-photon microscopy. Hence, we report the synthesis of 5 probes based on different donor cores (phenoxazine, acridane, phenazasiline and phenothiazine) and the study of their linear and non-linear photophysical properties. TD-DFT calculations were performed and were able to highlight the structure-property relationship of this series. All these studies highlight the great potential of three of these biocompatible dyes for two-Photon microscopy, as they both exhibit high two-photon cross-sections (up to 3 650 GM) and emit orange to red light. This potential was confirmed through live-cell two-photon microscopy experiments, leading to images with very high brightness and contrast.

Inhaled transfection particles have to penetrate the mucus layer lining the airways to successfully deliver their therapeutic nucleic acid payload to target cells in the underlying epithelium. However, the in vitro models used for evaluating gene carrier efficiency often disregard this viscous defensive barrier. In this study, the two mucussecreting cell lines NCI-H292 and Calu-3 were selected to develop a series of epithelial models displaying gradual mucus production. In NCI-H292 models, a gradual increase in the MUC5AC mucin was obtained after cell exposure to inducers. In Calu-3 models, MUC5AC production increased as a function of culture duration (3, 7, 14 days) at the air-liquid interface (ALI). Six DOPC-derived cationic lipids were designed and their pDNA delivery activity was evaluated to validate these cellular models. The strongest impairment of the lipid delivery activity was observed in the Calu-3 14-d ALI model. The MUC5AC production in this model was the greatest and the mucus layer was 20 µm thick. The mucus exhibited a solid viscoelastic behavior, and represented a major hindrance to lipoplex diffusion. The Calu-3 14-d ALI model will be highly useful for accurate evaluation of gene carriers intended for airway administration and characterization of their interactions with the mucus.

The use of caged compounds, defined as photolabile precursors of biological effectors, have proven to be attractive in various fields of biology. Photolytical reactions have been widely used to allow a rapid and efficient concentration jump of various biological effectors within organized biological systems. During the last two decades, the challenge was to overcome the difficulty that only high energy UV light was used to induce photochemical reactions on photoremovable protecting groups (PPGs). Infrared-sensitive PPGs should be able to improve in vivo applications of caged compounds. The present review is focused on recent strategies enabling the use of excitation wavelength inside the photo-therapeutical window (600–1000 nm) to efficiently and specifically cleave a chemical bond.

The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. Investigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells’ invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK–Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK–paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK–paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. In-vestigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells’ invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK–Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK–paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK–paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

Background: A positive surface charge has been largely associated with nanoparticle (NP) toxicity. However, by screening a carbon NP library in macrophages, we found that a cationic charge does not systematically translate into toxicity. To get deeper insight into this, we carried out a comprehensive study on 5 cationic carbon NPs (NP2 to NP6) exhibiting a similar zeta (ζ) potential value (from + 20.6 to + 26.9 mV) but displaying an increasing surface charge density (electrokinetic charge, Qek from 0.23 to 4.39 µmol/g). An anionic and non-cytotoxic NP (NP1, ζ-potential = − 38.5 mV) was used as control. Results: The 5 cationic NPs induced high (NP6 and NP5, Qek of 2.95 and 4.39 µmol/g, respectively), little (NP3 and NP4, Qek of 0.78 and 1.35 µmol/g, respectively) or no (NP2, Qek of 0.23 µmol/g) viability loss in THP-1-derived macrophages exposed for 24 h to escalating NP dose (3 to 200 µg/mL). A similar toxicity trend was observed in airway epithelial cells (A549 and Calu-3), with less viability loss than in THP-1 cells. NP3, NP5 and NP6 were taken up by THP-1 cells at 4 h, whereas NP1, NP2 and NP4 were not. Among the 6 NPs, only NP5 and NP6 with the highest surface charge density induced significant oxidative stress, IL-8 release, mitochondrial dysfunction and loss in lysosomal integrity in THP-1 cells. As well, in mice, NP5 and NP6 only induced airway inflammation. NP5 also increased allergen-induced immune response, airway inflammation and mucus production. Conclusions: Thus, this study clearly reveals that the surface charge density of a cationic carbon NP rather than the absolute value of its ζ-potential is a relevant descriptor of its in vitro and in vivo toxicity

In this study, we report the synthesis, linear and non-linear photophysical studies and live cell imaging of two two-photon activatable probes based on a silafluorene core: SiFluo-V and SiFluo-L. Thanks to their quadrupolar (A-π-D-π-A) design, these probes exhibit respectively good to impressive two-photon cross sections (from 210 GM to 2150 GM). TD-DFT calculations support the experimental evidence that SiFluo-L displays far better two-photon absorption properties than SiFluo-V. Moreover, SiFluo-L possesses all requirements for bioimaging as it is water soluble, cell-permeant and and presents a low cytotoxicity (IC80≥10 µM). It labels mitochondria in live-cell imaging at low laser power with high brightness, contrast and photostability. This study demonstrates that silafluorene is a promising core to develop new two-photon fluorophores for live cell imaging.

This study aimed at discriminating with sensitivity the toxicological effects of carbon dots (CDs) with various zeta potential (ζ) and charge density (Qek) in different cellular models of the human respiratory tract. One anionic and three cationic CDs were synthetized as follows: CD-COOH (ζ = −43.3 mV); CD-PEI600 (Qek = 4.70 µmol/mg; ζ = +31.8 mV); CD-PEHA (Qek = 3.30 µmol/mg; ζ = +29.2 mV) and CD-DMEDA (Qek = 0.01 µmol/mg; ζ = +11.1 mV). Epithelial cells (A549) and macrophages (THP-1) were seeded alone or as co-cultures with different A549:THP-1 ratios. The obtained models were characterized, and multiple biological responses evoked by CDs were assessed in the mono-cultures and the best co-culture model. With 14% macrophages, the 2:1 ratio co-culture best mimicked the in vivo conditions and responded to lipopolysaccharides. The anionic CD did not induce any effect in the mono-cultures nor in the co-culture. Among the cationic CDs, the one with the highest charge density (CD-PEI600) induced the most pronounced responses whatever the culture model. The cationic CDs of low charge density (CD-PEHA and CD-DMEDA) evoked similar responses in the mono-cultures, whereas in the co-culture, the three cationic CDs ranked according to their charge density (CD-PEI600 > CD-PEHA > CD-DMEDA), when taking into account their inflammatory effect. Thus, the co-culture system developed in this study appears to be a sensitive model for finely discriminating the toxicological profile of cationic nanoparticles differing by the density of their surface charges.

Alkylphospholipids (APLs) have elicited great interest as antitumor agents due to their unique mode of action on cell membranes. However, their clinical applications have been limited so far by high hemolytic activity. Recently, cationic prodrugs of erufosine, a most promising APL, have been shown to mediate efficient intracellular gene delivery, while preserving the antiproliferative properties of the parent APL. Here, cationic prodrugs of the two APLs that are currently used in the clinic, miltefosine, and perifosine, are investigated and compared to the erufosine prodrugs. Their synthesis, stability, gene delivery and self-assembly properties, and hemolytic activity are discussed in detail. Finally, the potential of the pro-miltefosine and pro-perifosine compounds M E12 and P E12 in combined antitumor therapy is demonstrated using pUNO1-hTRAIL, a plasmid DNA encoding TRAIL, a member of the TNF superfamily. With these pro-APL compounds, we provide a proof of concept for a new promising strategy for cancer therapy combining gene therapy and APL-based chemotherapy.

Hemolysis is a serious side effect of antitumor alkylphospholipids (APLs) that limits dose levels and is a constraint in their use in therapeutic regimen. Nine prodrugs of promising APLs (miltefosine, perifosine, and erufosine) were synthesized so as to decrease their membrane activity and improve their toxicity profile while preserving their antineoplastic potency. Methods The synthesis of the pro-APLs was straightforwardly achieved in one step starting from the parent APLs. The critical aggregation concentration of the prodrugs, their hydrolytic stability under various pH conditions, their blood compatibility and cytotoxicity in three different cell lines were determined and compared to those of the parent antitumor lipids. Results The APL prodrugs display antitumor activity which is similar to that of the parent alkylphospholipids but without associated hemolytic toxicity. Conclusion The pro-APL compounds may be considered as intravenously injectable derivatives of APLs. They could thus address one of the major issues met in cancer therapies involving antitumor lipids and restricting their utilization to oral and topical administration because of limited maximum tolerated dose.

Scaffold-assisted gene therapy is a highly promising tool to treat articular cartilage lesions upon direct delivery of chondrogenic candidate sequences. The goal of this study was to examine the feasibility and benefits of providing highly chondroreparative agents, the cartilage-specific sex-determining region Y-type high-mobility group 9 (SOX9) transcription factor or the transforming growth factor beta (TGF-β), to human bone marrow-derived mesenchymal stromal cells (hMSCs) via clinically adapted, independent recombinant adeno-associated virus (rAAV) vectors formulated with carbon dots (CDs), a novel class of carbon-dominated nanomaterials. Effective complexation and release of a reporter rAAV-lacZ vector was achieved using four different CDs elaborated from 1-citric acid and pentaethylenehexamine (CD-1); 2-citric acid, poly(ethylene glycol) monomethyl ether (MW 550 Da), and N,N-dimethylethylenediamine (CD-2); 3-citric acid, branched poly(ethylenimine) (MW 600 Da), and poly(ethylene glycol) monomethyl ether (MW 2 kDa) (CD-3); and 4-citric acid and branched poly(ethylenimine) (MW 600 Da) (CD-4), allowing for the genetic modification of hMSCs. Among the nanoparticles, CD-2 showed an optimal ability for rAAV delivery (up to 2.2-fold increase in lacZ expression relative to free vector treatment with 100% cell viability for at least 10 days, the longest time point examined). Administration of therapeutic (SOX9, TGF-β) rAAV vectors in hMSCs via CD-2 led to the effective overexpression of each independent transgene, promoting enhanced cell proliferation (TGF-β) and cartilage matrix deposition (glycosaminoglycans, type-II collagen) for at least 21 days relative to control treatments (CD-2 lacking rAAV or associated to rAAV-lacZ), while advantageously restricting undesirable type-I and -X collagen deposition. These results reveal the potential of CD-guided rAAV gene administration in hMSCs as safe, non-invasive systems for translational strategies to enhance cartilage repair. View Full-Text

The use of photolabile protecting groups (PPGs) has been growing in emphasis for decades, and they nowadays enable cutting-edge results in numerous fields ranging from organic synthesis to neurosciences. PPGs are chemical entities that can be conjugated to a biomolecule to hide its biological activity, forming a stable so called “caged compound”. This conjugate can be simply cleaved by light and therefore, the functionality of the biomolecule is restored with the formation of a PPG by-product. However, there is a sizeable need of PPGs able to quantify the “uncaging” process. In this review, we will discuss several strategies leading to an acute quantification of the uncaging events by fluorescence. In particular, we will focus on how molecular engineering of PPG could open new opportunities by an easy access to photoactivation protocols

Multilayered coated liposomes were prepared using the layer-by-layer (LbL) technique in an effort to improve their stability in biological media. The formulation strategy was based on the alternate deposition of two biocompatible and biodegradable polyelectrolytes – poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) – on negatively charged small unilamellar vesicles (SUVs). Some parameters of the formulation process were optimized such as the polyelectrolyte concentration and the purification procedure. This optimized procedure has allowed the development of very homogeneous formulations of liposomes coated with up to 6 layers of polymers (so-called layersomes). The coating was characterized by dynamic light scattering (DLS), zeta potential measurements and Förster resonance energy transfer (FRET) between two fluorescently labeled polyelectrolytes. Studies on the stability of the formulations at 4 °C in a buffered solution have shown that most structures are stable over 1 month without impacting their encapsulation capacity. In addition, fluorophore release experiments have demonstrated a better resistance of the layersomes in the presence of a non-ionic detergent (Triton™ X-100) as well as in the presence of phospholipase A2 and human plasma. In conclusion, new multilayered liposomes have been developed to increase the stability of conventional liposomes in biological environments.

With the aim of developing new molecular theranostic agents, a π-extended Zn(II) porphyrin as photosensitizer for photodynamic therapy (PDT) linked to two GdDOTA-type complexes for magnetic resonance imaging (MRI) detection was synthesized. The relaxivity studies revealed a much higher relaxivity value per Gd ion for this medium sized molecule (19.32 mM–1 s–1 at 20 MHz and 298 K) compared to clinical contrast agents—a value which strongly increases in the presence of bovine serum albumin, reaching 25.22 mM–1 s–1. Moreover, the photophysical studies showed the strong ability of the molecule to absorb light in the deep red (670 nm, ε ≈ 60000 M–1 cm–1) and in the near-infrared following two-photon excitation (920 nm, σ2 ≈ 650 GM). The conjugate is also able to generate singlet oxygen, with a quantum yield of 0.58 in DMSO. Promising results were obtained in cellular studies, demonstrating that the conjugate is internalized in HeLa cells at micromolar concentration and leads to 70% of cell death following 30 min irradiation at 660 nm. These results confirm the potential of the designed molecule as an imaging and therapeutic agent.

Sixteen cationic prodrugs of the antitumor alkylphospholipid (APL) erufosine were rationally synthesized to provide original gene delivery reagents with improved cytotoxicity profile. The DNA complexation properties of these cationic lipids were determined and associated transfection rates were measured. Furthermore, the self‐assembly properties of the pro‐erufosine compounds were investigated and their critical aggregation concentration was determined. Their hydrolytic stability under pH conditions mimicking the extracellular environment and the late endosome milieu was measured. Hemolytic activity and cytotoxicity of the compounds were investigated. The results obtained in various cell lines demonstrate that the prodrugs of erufosine display antineoplastic activity similar to that of the parent antitumor drug but are not associated with hemolytic toxicity, which is a dose‐limiting side effect of APLs and a major obstacle to their use in anticancer therapeutic regimen. Furthermore, by using lipoplexes prepared from a prodrug of erufosine and a plasmid DNA encoding a pro‐apoptotic protein (TRAIL), evidence was provided for selective cytotoxicity towards tumor cells while nontumor cells were resistant. This study demonstrates that the combination approach involving well tolerated erufosine cationic prodrugs and cancer gene therapy holds significant promise in tumor therapy.

Carbon dots (CDs) are emerging nanomaterial in medicine and pharmacy. To explore the impact of physicochemical characteristics on their safety, we synthesized a library of 35 CDs exhibiting different size, charge, chemical composition and surface coating, using various starting materials (carbon source and passivation reagent) and carbonization procedures. The 35 CDs triggered different levels of viability loss when incubated with human macrophages at 3-200 µg/mL for 24 h. The smaller NPs (10-20 nm) were more toxic that larger ones (40-100 nm), whereas NPs that aggregated in culture medium were more toxic than dispersed ones. A positive correlation was found between CD charge or nitrogen content and toxicity. Furthermore, a greater toxicity was observed for CDs prepared from high molecular weight polyamines, suggesting a role of the CD global density of positive charges, rather than the charge at the CD surface, in the CD toxicity. At last, PEG decoration decreased the toxicity of cationic NPs. In conclusion, the size, aggregation in culture medium, charge, nitrogen content, nature of the passivation agent and synthesis procedure were found to influence CD toxicity, making it difficult to predict CD safety from a single characteristic.

Cationic carbon dots (CDs) have been recently described as nucleic acid carriers with high in vitro and in vivo transfection efficiency and imaging properties. However, developing nanoparticles (NPs) for bio-medical applications requires assessing their safety. In the present study, we characterized the cell uptake and trafficking, as well as the cell viability loss, oxidative stress, inflammation, and mitochondrial and lysosomal perturbations evoked by cationic CDs prepared by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine (bPEI25k), using THP-1-derived macrophages. CDs were rapidly internalized by cells and addressed to the lysosomes after their cell entry. The NPs induced a dose-and time-dependent loss in cell viability that was associated with oxidative stress and IL-8 release. The CDs triggered also a dose-dependent loss in lysosome integrity , mitochondrial dysfunction, and NLRP3 inflammasome activation. Inhibition of the lysosomal protease cathepsin B significantly reduced CD-induced mitochondrial dysfunction and NLRP3 inflammasome activation, suggesting a pivotal role of the lysosome in the toxicological effects of the NPs. Our study provides for the first time a mechanistic pathway for the toxicological effects of bPEI25k-based cationic CDs.

Carbon dots (CDs) have been intensively investigated due to their unique photoluminescence (PL) properties that are improved through surface passivation with nitrogen-containing groups. Recently, gene delivery applications emerged as passivation of CDs may yield positively charged nanoparticles that can interact with negatively charged nucleic acids. However previous work in the field focused on the use of high molecular weight polyamines for CD passivation, posing the problem of the separation of nanoparticles from residual polymer that is harmful to cells. In this work, cationic CDs were prepared by pyrolysis of citric acid/bPEI600 (1/4, w/w) so unreacted low molecular weight reagents could be conveniently eliminated by extensive dialysis. Various reaction conditions and activation modes were evaluated and eleven CDs that exhibited superior solubility in water were produced. All the nanoparticles were characterized with respect to their physical, optical and PL properties and their ability to deliver plasmid DNA to mammal cells was evaluated. Despite their similar physical properties, the CDs displayed marked differences in their gene delivery efficiency. CDs produced under microwave irradiation in a domestic oven were revealed to be superior to all the other nanoparticles produced in this study and compared to the gold standard transfection reagent bPEI25k, with an optimal CD/pDNA w/w ratio that was significantly down shifted, as was the associated cytotoxicity.

Despite the advantages of photodynamic therapy (PDT) over chemotherapy or radiotherapy such as low side effects, lack of treatment resistance and spatial selectivity inherent to light activation of the drug, several limitations especially related to the photosensitiser (PS) prevent PDT from becoming widespread in oncology. Herein, new folic acid- and biotin-conjugated PSs for tumour-targeting PDT are reported, with promising properties related to PDT such as intense absorption following one-photon excitation in the red or two-photon excitation in the near-infrared, and also high singlet oxygen quantum yield (close to 70% in DMSO). Cellular studies demonstrated that both targeted PSs induced phototoxicity, the folate-targeted PS being the most effective one with 80% of cell death following 30 min of irradiation and a phototoxicity four times higher than that of the non-targeted PS. This result is in accordance with the uptake of the folate-targeted PS in HeLa cells, mediated by the folate receptors. Moreover, this folate-targeted PS was also phototoxic following two-photon excitation at 920 nm, opening new perspectives for highly selective PDT treatment of small and deep tumours.

A molecular theranostic agent designed for photodynamic therapy (PDT) treatment in the near-infrared and for imaging tissue tumors with magnetic resonance imaging (MRI) is reported. It consists of a linear pi-conjugated Zn(II) porphyrin dimer linked at each extremity to a GdDOTA-type complex. This agent has shown very promising potential for PDT applications with good singlet oxygen generation in DMSO and high linear absorption in the near- infrared (lambda(max) = 746 nm, epsilon approximate to 10(5) M-1 cm(-1)). Moreover, this molecule has a propensity for two-photon excited PDT with high two-photon cross sections (similar to 8000 GM in 880-930 nm range), which should allow for deeper tumor treatments and higher spatial precision as compared to conventional one-photon PDT. Regarding the MRI contrast agent properties, the molecule has shown superior relaxivity (14.4 mM(-1) s(-1) at 40 MHz, 298 K) in comparison to clinical contrast agents and the ability to be internalized in cells, thanks to its amphiphilic character. Irradiation of HeLa cells using either one-photon (740 nm) or two-photon excitation (910 nm) has led in both cases to important cell death.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that is overexpressed and activated in many human cancers. FAK transmits signals to a wide range of targets through both kinase-dependant and independent mechanism thereby playing essential roles in cell survival, proliferation, migration and invasion. In the past years, small molecules that inhibit FAK kinase function have been developed and show reduced cancer progression and metastasis in several preclinical models. Clinical trials have been conducted and these molecules display limited adverse effect in patients. FAK contain multiple functional domains and thus exhibit both important scaffolding functions. In this review, we describe the major FAK interactions relevant in cancer signalling and discuss how such knowledge provide rational for the development of Protein-Protein Interactions (PPI) inhibitors.

We report the synthesis and photolytic properties of caged 9aminodoxycycline derivatives modified with 2-{4'-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9aminodoxycycline showed complex photochemical reactions but did release 10% of 9-aminodoxycycline. However, 9-(PEG7DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells.

Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as N -methyl- d -glucamine (NMDG + ). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG + -permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG + permeability superimposes that of Na + and demonstrate that the molecular motions leading to the permeation of NMDG + are very similar to those that drive Na + flow. We found, however, that NMDG + “percolates” 10 times slower than Na + in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG + but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.

Three zwitterionic meso-substituted A(3)B- and AB(2)C-porphyrins containing one sulfonato alkylpyridinium substituent and three or two alkoxy-substituted phenyl groups were synthesized in good yield and fully characterized as to their physicochemical properties by a variety of techniques. This new series of inner salt donor-acceptor meso-substituted porphyrin derivatives were prepared for possible application as amphiphilic probes for membrane insertion in the area of combined second-harmonic and two-photon fluorescence cellular microscopy. To this end, the linear and nonlinear optical properties of the compounds were characterized, together with their electrochemical and spectroelectrochemical properties in non-aqueous media. The neutral design of such molecules enabled us to determine their second order non-linear properties, both by Electric Field Induced Second Harmonic Generation and Hyper-Rayleigh Scattering. Two-photon absorption cross sections of these dyes were also measured by the two-photon induced fluorescence method. The zwitterionic nature of the inner salt results in very specific solvent-dependent redox-properties, which could be rationalized in terms of solvent-dependent ion-pairing. The overall data electrochemical and photophysical data indicates that these new porphyrinic systems should be good probes for membrane potential sensing.

We successfully introduced two‐photon‐sensitive photolabile groups ([7‐(diethylamino)coumarin‐4‐yl]methyl and p ‐dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three‐dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double‐strand displacement in a hydrogel and in neurons. Orthogonal two‐photon uncaging of the two cages is possible, thus enabling complex scenarios of three‐dimensional control of hybridization with light.

A molecular theranostic agent for magnetic resonance imaging (MRI) and photodynamic therapy (PDT) consisting of four [GdDTTA]− complexes (DTTA4− = diethylenetriamine-N,N,N″,N″-tetraacetate) linked to a meso-tetraphenylporphyrin core, as well as its yttrium(III) analogue, was synthesized. A variety of physicochemical methods were used to characterize the gadolinium(III) conjugate 1 both as an MRI contrast agent and as a photosensitizer. The proton relaxivity measured in H2O at 20 MHz and 25 °C, r1 = 43.7 mmol–1 s–1 per gadolinium center, is the highest reported for a bishydrated gadolinium(III)-based contrast agent of medium size and can be related to the rigidity of the molecule. The complex displays also a remarkable singlet oxygen quantum yield of ϕΔ = 0.45 in H2O, similar to that of a meso-tetrasulfonated porphyrin. We also evidenced the ability of the gadolinium(III) conjugate to penetrate in cancer cells with low cytotoxicity. Its phototoxicity on Hela cells was evaluated following incubation at low micromolar concentration and moderate light irradiation (21 J cm–2) induced 50% of cell death. Altogether, these results demonstrate the high potential of this conjugate as a theranostic agent for MRI and PDT.

A large number of plants used in traditional medicines have been shown to possess antitumor activities. The aims of this study were to evaluate any anticancer effect of the essential oil (EO) extracted from P. tortuosus against B16F10 melanoma cancer cells in vitro as well as in vivo. In vitro, EO was shown to induce apoptosis and to inhibit migration and invasion processes. Further investigation revealed that EO decreased focal adhesion and invadopodia formation which was accompanied by a drastic downregulation of FAK, Src, ERK, p130Cas and paxillin. Moreover, EO treatment decreased the expression level of p190RhoGAP, and Grb2, which impair cell migration and actin assembly. Mice bearing the melanoma cells were used to confirm any in vivo effectiveness of the EO as an anti-tumor promoting agent. In mice dosed with 100 mg EO/kg/d (for 27 days), tumor weight was inhibited by 98% compared to that in mice that did not receive the product. In conclusion, these data suggested to us that an EO of P. tortuosus could evolve to be a potential medicinal resource for use in the treatment of cancers.

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

The convergent synthesis and characterization of a potential theranostic agent, [DPP-ZnP-GdDOTA]-, which combines a diketopyrrolopyrrole-porphyrin component DPPZnP as a two-photon photosensitizer for photodynamic therapy (PDT) with a gadolinium(III) DOTA complex as a magnetic resonance imaging probe, is presented. [DPP-ZnP-GdDOTA]- has a remarkably high longitudinal water proton relaxivity(19.94 mm-1 s-1 at 20 MHz and 258C) for a monohydrated molecular system of this size. The Nuclear Magnetic Relaxation Dispersion (NMRD) profile is characteristic of slow rotation, related to the extended and rigid aromatic units integrated in the molecule and to self-aggregation occurring in aqueous solution. The two-photon properties were examinedand large two-photon absorption cross-sections around 1000 GM were determined between 910 and 940 nm in DCM with 1% pyridine and in DMSO. Furthermore, the newconjugate was able to generate singlet oxygen, with quantumyield of 0.42 and 0.68 in DCM with 1% pyridine and DMSO, respectively. Cellular studies were also performed. The [DPP-ZnP-GdDOTA]- conjugate demonstrated low dark toxicity and was able to induce high one-photon and moderate two-photon phototoxicity on cancer cells.

Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent plc value which may be as large as two pK(a) units and cannot be satisfactorily predicted.

Abstract Herein the synthesis, spectroscopic characterization, two‐photon absorption and electrochemical properties of 3,6‐disubstituted carbazole tweezers is reported. A dimer resulting from a Glaser homocoupling was isolated during a Sonogashira coupling reaction between a diethynyl‐carbazole spacer and a 5‐bromo‐triarylporphyrin and the properties of this original compound were compared with the 3,6‐disubstituted carbazole bisporphyrin tweezers. The dyads reported herein present a two‐photon absorption maximum at 920 nm with two‐photon absorption cross‐section in the 1200 GM range. Despite a strong linear absorption in the Soret region and moderate fluorescence quantum yield, they both lead to a high brightness reaching 30 000 M −1 cm −1 .

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20-30 % yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo and biocompatibility.

Polyurethane (PU) monomer mixtures containing commercially available o ‐nitrobenzyl‐based photocleavable monomers have been formulated and tested as low‐cost positive tone photoresists. The photolysis reaction is studied by UV spectroscopy. Well‐defined micropatterns on 2 μm thick photodegradable PU films are obtained using 365 nm light exposure. This strategy is also extended to improved formulations based on synthesized o ‐nitrobiphenylpropyl derivatives with enhanced photochemical properties for single photon excitation and high two‐photon absorption cross‐sections. Improved pattern resolution in 2D and the capability of 3D resolution using a scanning laser at 780 nm is demonstrated. This work demonstrates the potential of PUs as readily available, versatile, and easy‐to‐use photoresist materials for low‐cost lithography applications. image

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK(Y397F) reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.

Abstract We report the synthesis and photolytic properties of caged inorganic phosphates (Pi compounds) based on the 2‐(4′‐{bis[2‐(2‐methoxyethoxy)ethyl]amino}‐4‐nitro‐[1,1′‐biphenyl]‐3‐yl)propan‐1‐ol (EANBP) and 7‐(diethylamino)coumarin‐4‐yl]methyl (DEACM) protecting groups. The EANBP‐Pi showed unprecedented photolysis efficiency at 405 nm, with 95 % release of free phosphate and a quantum yield of 0.28. Thanks to the high two‐photon sensitivity of the EANBP chromophore, Pi release through two‐photon photolysis is also possible, with an action cross section of 20.5 GM at 800 nm. Two bioactivatable acetoxymethyl protection groups were added to the “caged‐Pi” compounds. The resulting triesters of phosphoric acid were able to diffuse through the cellular membranes of plant cells. Once inside a cell, the cleavage of these biocleavable motifs by intracellular esterases allows intracellular accumulation of EANBP‐Pi. Bis(AM)‐EANBP‐Pi therefore represents a very attractive tool for study of the Pi signal transduction cascade in living cells.

We propose an experimental approach, alternative to Langmuir trough, pendant or sessile drop experiments, to study insoluble monolayers at the air-water interface. The method is based on the direct deposition of an insoluble surfactant at the interface of an air bubble, measuring the surface tension according to the classical axisymmetric drop shape analysis (ADSA) technique. This bubble configuration, in contrast to the classical ones for studying Langmuir monolayers have several remarkable advantages like the easy control of the surrounding bulk composition (opening new potential research applications), the fast experimental time for a monolayer to be ready (&lt;20 min), the small bulk volume (10 mL), and mostly the simple way to carry out dilatational rheology. The experiment consists of performing compression of an insoluble monolayer recording the Π-AB curve (Π is the interfacial pressure and AB the bubble area) and obtaining dilatational rheology over the compression range establishing the E-Π curve (with E is the elastic modulus). We showed that the experimental results can be satisfactorily fitted using the Volmer's equation of state including the two-dimensional compressibility factor ε, offering access to the excluded area per molecule ω0 and to the number N of molecules at the interface, without initially knowing the amount of deposited material. This proof of concept study was carried out on dioleoyl-sn-glycerophosphatidylcholine (DOPC), dipalmitoyl-sn-glycerophosphatidylcholine (DPPC), and cholesterol at 20 °C, systems chosen to show qualitative differences in their thermodynamic behavior upon monolayer compression. Likewise, dilatational rheology of these insoluble monolayers allows evidencing the compressibility of the DOPC monolayer in contrast to the DPPC monolayer, and finally, the compression domains where the interface loses the surfactant through a comparison of the dilatational elasticity with the Gibbs elasticity calculated from the compression curves. Finally, we propose an example of the new application offered by the possibility to exchange the fluid phase surrounding the bubble, herein to study mixed monolayers made with soluble/insoluble surfactants (Tween 80/Span 65).

Five new non-ionic water-soluble two-photon dyes for fluorescence microscopy built around a diketopyrrolopyrrole central core were designed, prepared and characterized by 1H and 13C NMR, UV-Visible spectroscopy, HRMS and IR. Excitation and emission wavelengths were tuned by modifying the lengths of the conjugated systems and the nature of the substituents surrounding the diketopyrrolopyrrole central core. These fluorescent dyes are highly photostable and present high one- and two-photon brightness. Their application in confocal and two-photon excited fluorescence microscopy was performed on HeLa cell cultures with low power excitation.

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In the present work, we elaborated a synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), as a vehicle to study the lung toxicity of pristine multi-walled carbon nanotubes (MWCNT). MWCNT were dispersed in surfactant, saline or saline containing DPPC, BSA, Pluronic(®) F68 or sodium dodecyl sulfate, for comparison. Dispersions were characterized visually, and by light microscopy, dynamic light scattering and transmission electronic microscopy (TEM). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice upon single or repeated administrations was analyzed by histology and TEM. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF)-β1 and collagen, and by histology. We found that the elaborated surfactant is more effective in dispersing MWCNT when compared to the other agents, while being biocompatible. Surfactant-dispersed MWCNT distributed all throughout the mouse airways upon single and repeated administrations and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. Mice that received a single administration of MWCNT showed neutrophil infiltrate and greater concentrations of tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC) and interleukin (IL)-17 in BALF when compared to controls. After repeated MWCNT administrations, increases in macrophage number, KC and TGF-β1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed. Altogether, the elaborated lung surfactant could be a valuable tool to further study the toxicological impact of pristine MWCNT in laboratory animals.

CD47 is a ubiquitous 50 kDa five-spanning membrane receptor that belongs to the immunoglobulin superfamily. This receptor, also known as integrin-associated protein, mediates cell-to-cell communication by ligation to transmembrane signal-regulatory proteins SIRPalpha and SIRPgamma and interacts with integrins. CD47 is also implicated in cell-extracellular matrix interactions via ligation with thrombospondins. Furthermore, CD47 is involved in many and diverse cellular processes, including apoptosis, proliferation, adhesion and migration. It also plays a key role in many immune and cardiovascular responses. Thus, this multifaceted receptor might be a central actor in the tumour microenvironment. Solid tumours are composed of not only cancer cells that actively proliferate but also other cell types including immune cells and fibroblasts that make up the tumour microenvironment. Tumour cell proliferation is strongly sustained by continuous sprouting of new vessels, which also represents a gate for metastasis. Moreover, infiltration of inflammatory cells is observed in most neoplasms. Much evidence has accumulated indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that compose the tumour microenvironment, targeting CD47 could represent an original therapeutic strategy in the field of oncology. We present a current overview of the biological effects associated with CD47 on cancer cells and stromal cells.

The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent “tethering” reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

BACKGROUND AND PURPOSE Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA A receptors (GABA A Rs). We previously reported that trans ‐6,4′‐dimethoxyretrochalcone (Rc‐OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA A Rs. Here, we evaluate the functional modulations of Rc‐OMe, along with two other synthetic derivatives trans ‐6‐bromo‐4′‐methoxyretrochalcone (Rc‐Br) and 4,3′‐dimethoxychalcone (Ch‐OMe) on GABA A Rs. EXPERIMENTAL APPROACH Whole‐cell patch‐clamp recordings were made to determine the effects of these derivatives on GABA A Rs expressed in HEK‐293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain. KEY RESULTS Rc‐OMe strongly potentiated GABA‐evoked currents at recombinant α 1–4 β 2 γ 2s and α 4 β 3 δ receptors but much less at α 1 β 2 and α 4 β 3 . Rc‐Br and Ch‐OMe potentiated GABA‐evoked currents at α 1 β 2 γ 2s . The potentiation by Rc‐OMe was only reduced at α 1 H101Rβ 2 γ 2s and α 1 β 2 N265Sγ 2s , mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc‐OMe and pentobarbital as well as by Rc‐OMe and the neurosteroid 3α,21‐dihydroxy‐5α‐pregnan‐20‐one was supra‐additive. Rc‐OMe modulation exhibited no apparent voltage‐dependence, but was markedly dependent on GABA concentration. In neurones, Rc‐Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc‐OMe and Rc‐Br positively modulated synaptic and extrasynaptic diazepam‐insensitive GABA A Rs. CONCLUSIONS AND IMPLICATIONS The trans ‐retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA A Rs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA A ‐related disorders in vivo .

The search for chemical probes which allow a controlled fluorescence activation in living cells represent a major challenge in chemical biology. To be useful, such probes have to be specifically targeted to cellular proteins allowing thereof the analysis of dynamic aspects of this protein in its cellular environment. The present paper describes different methods which have been developed to control cellular fluorescence activation emphasizing the photochemical activation methods known to be orthogonal to most cellular components and, in addition, allowing a spatio-temporal controlled triggering of the fluorescent signal.

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α+ and CD8α− cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4− and CD4+ CD8α− cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4− and CD4+ cDC are equivalent in their capacity to prime and direct CD4+ and CD8+ T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4− and CD4+ cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4+ counterparts, CD4− cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4+ and CD4− cDC subsets that may be important in immune responses.

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.

Two-photon (TP) excitation of organic chromophores is of great interest for decades, as applications of such phenomena from 3-dimentional (3D) microfabrication to optical limiting and optical data storage, are of increasing importance. More recently, two-photon excitation found important applications in biology, notably in two-photon excited microscopy (TPEM) or two-photon photodynamic therapy (2P-PDT). Nevertheless, these techniques were using dyes or sensitizers designed for one-photon processes with low two-photon response. The lack of efficient molecules specifically designed for two-photon applications has led us to design new chromophores for biological applications with increased sensitivity to two-photon excitation and specifically added properties useful in biological media, such as water solubility. Here we describe the molecular engineering of such dyes mainly for cell and small animal observation by TPEM and their conjugation to magnetic nanoparticles and bio-nanoparticles such as viruses.We will then focus on the possibility to use photochemical reaction for cell triggering by two-photon photorelease of biologically active substances the so-called two-photon uncaging.

Two-photon (TP) excitation of organic chromophores is of great interest for decades, as applications of such phenomena from 3-dimentional (3D) microfabrication to optical limiting and optical data storage, are of increasing importance. More recently, two-photon excitation found important applications in biology, notably in two-photon excited microscopy (TPEM) or two-photon photodynamic therapy (2P-PDT). Nevertheless, these techniques were using dyes or sensitizers designed for one-photon processes with low two-photon response. The lack of efficient molecules specifically designed for two-photon applications has led us to design new chromophores for biological applications with increased sensitivity to twophoton excitation and specifically added properties useful in biological media, such as water solubility. Here we describe the molecular engineering of such dyes mainly for cell and small animal observation by TPEM and their conjugation to magnetic nanoparticles and bio-nanoparticles such as viruses. We will then focus on the possibility to use photochemical reaction for cell triggering by two-photon photorelease of biologically active substances the so-called two-photon uncaging.

BACKGROUND AND PURPOSE Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells. EXPERIMENTAL APPROACHES Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured. KEY RESULTS Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G(i)-proteins and intracellular Ca2+ increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls. CONCLUSIONS AND IMPLICATIONS Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.

In order to have short photo-cleavable crosslinkers more suitable for the intramolecular crosslinking; we have synthesized homo-bifunctional photo-cleavable sulfhydryl groups crosslinkers that represent distances of 2.6–5.4 Å between the two reactive sites. These chemical probes were able to react with two equivalents of cysteines, and to photo-release them very efficiently by near-UV irradiation.

Light‐responsive biologically active compounds offer the possibility to study the dynamics of biological processes. Phototriggers and photoswitches have been designed, providing the capability to rapidly cause the initiation of wide range of dynamic biological phenomena. We will discuss, in this article, recent developments in the field of light‐triggered chemical tools, specially how two‐photon excitation, “caged” fluorophores, and the photoregulation of protein activities in combination with time‐resolved x‐ray techniques should break new grounds in the understanding of dynamic biological processes.

We report herein the synthesis and the development of homo-bifunctional photo-cleavable sulfhydryl group cross-linkers that are able to react and then to photorelease two cysteines leading to the photoregulation of cross-linkers cleavage.

This method aims at providing structural information on protein or nucleoprotein complexes by high-resolution electron microscopy. The objective is to promote the self-assembly of the macromolecules into two-dimensional crystals in order to use electron crystallography methods. When combined with observations in the frozen hydrated states and dedicated image processing software these methods can provide detailed 3-D models of the complex. The 2-D crystals of soluble nucleoprotein complexes are formed on lipid monolayers spread at the air-water interface. The macromolecule of interest is targeted to the monolayer by either electrostatic or ligand-induced interactions with the hydrophilic head group of the lipid. Upon interaction with the lipids, the nucleoprotein complex is concentrated at the vicinity of the lipid layer whose in-plane mobility facilitates the contacts between macromolecules and the formation of ordered arrays.

This method of two-dimensional crystallization of proteins on a lipid monolayer aims at producing 2D crystals of membrane proteins, which can provide structural information at high resolution by electron crystallography. A lipid monolayer is spread over the whole air–water interface of a drop, which provides a substrate for protein binding. The protein of interest is then adsorbed onto the lipid monolayer and forms a closely packed layer. The reconstitution step of the membrane protein into a lipid bilayer is realized by elimination of detergent. The combined effect of the elevated protein concentration, the alignment of the protein on the lipid monolayer, and the fluid monolayer film properties are conducive, in some cases, to the formation of 2D crystals. The use of locally fluorinated lipids in avoiding solubilization of the lipid monolayer by detergents is presented and discussed.

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