A Conjugate between Lqh-8/6, a Natural Peptide Analogue of Chlorotoxin, and Doxorubicin Efficiently Induces Glioma Cell Death.

Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.

Titanium dioxide particles from the diet: involvement in the genesis of inflammatory bowel diseases and colorectal cancer.

The gastrointestinal tract is a complex interface between the external environment and the immune system. Its ability to control uptake across the mucosa and to protect the body from damage of harmful substances from the lumen is defined as the intestinal barrier function (IBF). The IBF involves four elements: the intestinal microbiota, the mucus layer, the epithelium and the immune system. Its dysfunction is linked with human diseases including inflammatory, metabolic, infectious, autoimmune and neurologic disorders. Most of these diseases are complex and involve genetic, psychological and environmental factors. Over the past 10 years, many genetic polymorphisms predisposing to inflammatory bowel disease (IBD) have been identified. Yet, it is now clear that they are insufficient to explain the onset of these chronic diseases. Although it has been evidenced that some environmental factors such as cigarette smoking or carbohydrate intake are associated with IBD, other environmental factors also present potential health risks such as ingestion of food additives introduced in the human diet, including those composed of mineral particles, by altering the four elements of the intestinal barrier function. The aim of this review is to provide a critical opinion on the potential of TiO2 particles, especially when used as a food additive, to alter the four elements of the intestinal barrier function, and consequently to evaluate if this additive would likely play a role in the development and/or exacerbation of IBD.

Toxicological impact of acute exposure to E171 food additive and TiO2 nanoparticles on a co-culture of Caco-2 and HT29-MTX intestinal cells.

TiO2 particles are widely used in products for everyday consumption, such as cosmetics and food; their possible adverse effects on human health must therefore be investigated. The aim of this study was to document in vitro impact of the food additive E171, i.e. TiO2, and of TiO2 nanoparticles, on a co-culture of Caco-2 and HT29-MTX cells, which is an in vitro model for human intestine. Cells were exposed to TiO2 particles three days after seeding, i.e. while they were not fully differentiated. Cell viability, reactive oxygen species (ROS) levels and DNA integrity were assessed, by MTT assay, DCFH-DA assay, alkaline and Fpg-modified comet assay and 8-oxo-dGuo measurement by HPLC-MS/MS. The mRNA expression of genes involved in ROS regulation, DNA repair via base-excision repair, and endoplasmic reticulum stress was assessed by RT-qPCR. Exposure to TiO2 particles resulted in increased intracellular ROS levels, but did not impair cell viability and did not cause any oxidative damage to DNA. Only minor changes in mRNA expression were detected. Altogether, this shows that E171 food additive and TiO2 nanoparticles only produce minor effects to this in vitro intestinal cell model.

H-Rubies, a new family of red emitting fluorescent pH sensors for living cells.

Monitoring intracellular pH has drawn much attention due to its undeniably important function in cells. The widespread development of fluorescent imaging techniques makes pH sensitive fluorescent dyes valuable tools, especially red-emitting dyes which help to avoid the overcrowded green end of the spectral band. Herein, we present H-Rubies, a family of pH sensors based on a phenol moiety and a X-rhodamine fluorophore that display a bright red fluorescence upon acidification with pKa values spanning from 4 to 9. Slight structural modifications led to dramatic changes in their physicochemical properties and a relationship between their structures, their ability to form H-aggregates, and their apparent pKa was established. While molecular form H-Rubies can be used to monitor mitochondrial acidification of glioma cells, their functionalised forms were linked via click chemistry to dextrans or microbeads containing a near infrared Cy5 (Alexa-647) in order to provide ratiometric systems that were used to measure respectively the phagosomal and endosomal pH in macrophages (RAW 264.7 cells) using flow cytometry.

Cell-penetrating nanobiosensors for pointillistic intracellular Ca2+-transient detection.

Small-molecule chemical calcium (Ca(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but their intracellular use remains a major challenge. We designed cell-penetrating FRET-based Ca(2+) nanobiosensors for the detection of local Ca(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca(2+) indicator, as an acceptor. With Ca(2+)-binding affinities covering the range of 3-20 µM, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-d-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca(2+) transients following agonist application when imaged by TIRF microscopy. Our work presents the elaboration of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca(2+) transients.

Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-L-maurocalcine analog.

L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)µl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Cell penetration properties of a highly efficient mini maurocalcine Peptide.

Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin's hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments.

Small efficient cell-penetrating peptides derived from scorpion toxin maurocalcine.

Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.

Cell-permeable Ln(III) chelate-functionalized InP quantum dots as multimodal imaging agents.

Quantum dots (QDs) are ideal scaffolds for the development of multimodal imaging agents, but their application in clinical diagnostics is limited by the toxicity of classical CdSe QDs. A new bimodal MRI/optical nanosized contrast agent with high gadolinium payload has been prepared through direct covalent attachment of up to 80 Gd(III) chelates on fluorescent nontoxic InP/ZnS QDs. It shows a high relaxivity of 900 mM(-1) s(-1) (13 mM(-1 )s(-1) per Gd ion) at 35 MHz (0.81 T) and 298 K, while the bright luminescence of the QDs is preserved. Eu(III) and Tb(III) chelates were also successfully grafted to the InP/ZnS QDs. The absence of energy transfer between the QD and lanthanide emitting centers results in a multicolor system. Using this convenient direct grafting strategy additional targeting ligands can be included on the QD. Here a cell-penetrating peptide has been co-grafted in a one-pot reaction to afford a cell-permeable multimodal multimeric MRI contrast agent that reports cellular localization by fluorescence and provides high relaxivity and increased tissue retention with respect to commercial contrast agents.