Cell Nucleus-Targeting Zwitterionic Carbon Dots

An innovative nucleus-targeting zwitterionic carbon dot (CD) vehicle has been developed for anticancer drug delivery and optical monitoring. The zwitterionic functional groups of the CDs introduced by a simple one-step synthesis using beta-alanine as a passivating and zwitterionic ligand allow cytoplasmic uptake and subsequent nuclear translocation of the CDs. Moreover, multicolor fluorescence improves the accuracy of the CDs as an optical code. The CD-based drug delivery system constructed by non-covalent grafting of doxorubicin, exhibits superior antitumor efficacy owing to enhanced nuclear delivery in vitro and tumor accumulation in vivo, resulting in highly effective tumor growth inhibition. Since the zwitterionic CDs are highly biocompatible and effectively translocated into the nucleus, it provides a compelling solution to a multifunctional nanoparticle for substantially enhanced nuclear uptake of drugs and optical monitoring of translocation.open

Stable, Precise, and Reproducible Patterning of Bicoid and Hunchback Molecules in the Early Drosophila Embryo

Precise patterning of morphogen molecules and their accurate reading out are of key importance in embryonic development. Recent experiments have visualized distributions of proteins in developing embryos and shown that the gradient of concentration of Bicoid morphogen in Drosophila embryos is established rapidly after fertilization and remains stable through syncytial mitoses. This stable Bicoid gradient is read out in a precise way to distribute Hunchback with small fluctuations in each embryo and in a reproducible way, with small embryo-to-embryo fluctuation. The mechanisms of such stable, precise, and reproducible patterning through noisy cellular processes, however, still remain mysterious. To address these issues, here we develop the one- and three-dimensional stochastic models of the early Drosophila embryo. The simulated results show that the fluctuation in expression of the hunchback gene is dominated by the random arrival of Bicoid at the hunchback enhancer. Slow diffusion of Hunchback protein, however, averages out this intense fluctuation, leading to the precise patterning of distribution of Hunchback without loss of sharpness of the boundary of its distribution. The coordinated rates of diffusion and transport of input Bicoid and output Hunchback play decisive roles in suppressing fluctuations arising from the dynamical structure change in embryos and those arising from the random diffusion of molecules, and give rise to the stable, precise, and reproducible patterning of Bicoid and Hunchback distributions

Control of Alzheimer's Amyloid Beta Toxicity by the High Molecular Weight Immunophilin FKBP52 and Copper Homeostasis in Drosophila

FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Aβ toxicity. Towards this goal, we generated Aβ transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Aβ and increased lifespan in Aβ flies, whereas loss of function of FKBP52 exacerbated these Aβ phenotypes. Interestingly, the Aβ pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (−/−) cells have increased intracellular copper and higher levels of Aβ. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Aβ peptides

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

<p>Abstract</p> <p>Background</p> <p>Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.</p> <p>Results</p> <p>Reaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated.</p> <p>Conclusions</p> <p>Multi-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to specific subcellular regions additional mechanisms must exist that limit or spatially coordinate caspase activation and/or protect diffusing soluble caspase substrates from unwanted proteolysis.</p

Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells

Aggregation and cytotoxicity of mutant proteins containing an expanded number of polyglutamine (polyQ) repeats is a hallmark of several diseases, including Huntington's disease (HD). Within cells, mutant Huntingtin (mHtt) and other polyglutamine expansion mutant proteins exist as monomers, soluble oligomers, and insoluble inclusion bodies (IBs). Determining which of these forms constitute a toxic species has proven difficult. Recent studies support a role for IBs as a cellular coping mechanism to sequester levels of potentially toxic soluble monomeric and oligomeric species of mHtt.When fused to a fluorescent reporter (GFP) and expressed in cells, the soluble monomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Htt(ex1)) and monitor their fates in live cells. Photobleaching analyses revealed a significant reduction in the mobilities of mHtt(ex1) variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtype and mHtt(ex1) formed oligomers, as evidenced by the formation of a fluorescent reporter. Only the mHtt(ex1) split-GFP oligomers assembled into IBs. Both FRAP and split-GFP approaches confirmed the ability of mHtt(ex1) to bind and incorporate wildtype Htt into soluble oligomers. We exploited the irreversible binding of split-GFP fragments to forcibly increase levels of soluble oligomeric mHtt(ex1). A corresponding increase in the rate of IBs formation and the number formed was observed. Importantly, higher levels of soluble mHtt(ex1) oligomers significantly correlated with increased mutant cytotoxicity, independent of the presence of IBs.Our study describes powerful and sensitive tools for investigating soluble oligomeric forms of expanded polyglutamine proteins, and their impact on cell viability. Moreover, these methods should be applicable for the detection of soluble oligomers of a wide variety of aggregation prone proteins

Magnetoliposome for alendronate delivery

1 - ArticleEncapsulation into liposomes has been developed in order to allow protection of therapeutical agents against enzymatic degradation, and to reduce doses and toxic side effects. Selective, targeted and controlled release of the drug out of the lipid vesicle is still, however, difficult to achieve. Meanwhile, thanks to their magnetic properties, superparamagnetic iron oxide (SPIO) nanoparticles have also been considered as good delivery vehicles after grafting a therapeutic drug on their surface. A combination of both properties (magnetic targeting and drug encapsulation) is evaluated to deliver an anticancer drug : alendronate, an hydroxymethylene bisphosphonate molecule. gamma-Fe(2)O(3) nanocrystals grafted with alendronate were tested with or without liposome encapsulation, with and without magnetic field, on three human cancer cell lines, MDA-MB231, A431 and U87-MG. Cytotoxicity was measured as well as drug internalization. While results were not identical on the three cell lines with the different formulations, an effective 100% cytotoxic effect could only be achieved with alendronate grafted-SPIO entrapped into liposomes and exposed to a magnetic field

In vitro assessment of liposomal neridronate on MDA-MB-231 human breast cancer cells

1 - ArticleBisphosphonates have been used for decades in the standard therapy of bone-related diseases, including bone metastasis of various malignancies, and they might as well be toxic on early cancer cells themselves. In order to allow a better delivery of neridronate (a N-containing bisphosphonate with relatively poor activity), liposomes were evaluated in vitro on cancer cell lines (MDA-MB 231, U87-MG and Caco2). After chemical synthesis, this water-soluble molecule was encapsulated into liposomes containing DOPC:DOPG:Chol (72:27:1 molar ratio). The influence of neridronate (free or liposomal) on cell viability or proliferation after treatment was evaluated using the MTT method, as well as cell migration and invasion assays; these techniques showed a drastic improvement of the action of neridronate on MDA-MB-231 cells with an EC50 fifty times lower when neridronate was encapsulated. Internalization of liposomes was followed by flow cytometry and fluorescence microscopy, demonstrating internalization via the endocytic pathway. Furthermore, since over-expression of matrix metalloproteinases (particularly MMP-2 and MMP-9) has been correlated to poor prognosis in many cancer types, detection of MMP expression is a satisfactory indication of the therapy efficiency and was then performed on treated cells. On MDA-MB-231 cells, MPPs expression was also significantly reduced by neridronate while entrapped in liposomes

Study of the intracellular nanoparticle-based radiosensitization mechanisms in F98 glioma cells treated with charged particle therapy through synchrotron-based infrared microspectroscopy

International audienceThe use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently,the use of NP has been extended to charged particle therapy in order to improve the performance inradioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved inNP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-basedFourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NPinduced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped withAuNP and GdNP were irradiated using several types of medical ion beams (proton, helium, carbon andoxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectralregions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellularmodifications were detected, such as conformational changes in secondary protein structures, intensityvariations in the lipid CHx stretching bands, as well as complex DNA rearrangements following chargedparticle therapy irradiations. These spectral features seem to be correlated with the already shownenhancement both in the DNA damage response and in the reactive oxygen species (ROS) production bythe NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational featureswere NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided newinsights into the molecular changes in response to NP-based RT treatments using ion beams, and highlighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to innovative radiotherapy approaches

New multimodal iron oxide nanoparticles as nanotools for cancer

9 - Conference Paper: NSTI Nanotech 2010: Bio Sensors, Instruments, Medical, Environment and Energy, 21-24 juin 2010, Anaheim (USA)The goal of this project is to elaborate new multifunctional magnetic nanovectors to vectorize biological interest molecules for therapy and diagnostic applications. Indeed, iron oxide nanoparticles, tanks to their magnetic properties, are used as contrast agents for MRI. Moreover the specific surface coating by interest molecules permit to consider them as a drug delivery vehicle for therapeutic molecules. Liposomes are also considering as good candidates for drug delivery systems. Indeed, lipid vesicle encapsulation allows protection of the agent against enzymatic degradation, and reduces the dose and toxics effects. However, the selective and controlled release of the drug out of the vesicles is still difficult to master, especially at cell and tissue levels. This can be achieved by magnetic targeting. Recent studies have shown that in addition to inhibit bone resorption, Hydroxymethylene Bisphosphonates molecules present anti-tumour properties in variety of cancer. However, these properties can not be exploited due to their very high affinity to bone. In order to overcome this problem, we use two strategies. The first one is the vectorization trough iron oxide nanocrystal surface functionalization. The second one was to use superparamagnetic liposomes