Hybrid Peptide-Alkoxyamine Drugs: A Strategy for the Development of a New Family of Antiplasmodial Drugs.

The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite's food vacuoles, our approach is summarized as "to dig its grave with its fork". However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model.

The epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach.

A protein nanocontainer targeting epithelial cancers: rational engineering, biochemical characterization, drug loading and cell delivery.

The development of drug delivery and imaging tools is a major challenge in human health, in particular in cancer pathologies. This work describes the optimization of a protein nanocontainer, belonging to the lectin protein family, for its use in epithelial cancer diagnosis and treatment. Indeed, it specifically targets a glycosidic marker, the T antigen, which is known to be characteristic of epithelial cancers. Its quaternary structure reveals a large hydrated inner cavity able to transport small therapeutic molecules. Optimization of the nanocontainer by site directed mutagenesis allowed controlling loading and release of confined drugs. Doxorubicin confinement was followed, both theoretically and experimentally, and provided a proof of concept for the use of this nanocontainer as a vectorization system. In OVCAR-3 cells, a human ovarian adenocarcinoma cell line that expresses the T antigen, the drug was observed to be delivered inside late endosomes/lysosomes. These results show that this new type of vectorization and imaging device opens new exciting perspectives in nano-theranostic approaches.

A Comparative Study on the Effects of Millisecond- and Microsecond-Pulsed Electric Field Treatments on the Permeabilization and Extraction of Pigments from Chlorella vulgaris.

The interdependencies of the two main processing parameters affecting "electroporation" (electric field strength and pulse duration) while using pulse duration in the range of milliseconds and microseconds on the permeabilization, inactivation, and extraction of pigments from Chlorella vulgaris was compared. While irreversible "electroporation" was observed above 4 kV/cm in the millisecond range, electric field strengths of ≥10 kV/cm were required in the microseconds range. However, to cause the electroporation of most of the 90 % of the population of C. vulgaris in the millisecond (5 kV/cm, 20 pulses) or microsecond (15 kV/cm, 25 pulses) range, the specific energy that was delivered was lower for microsecond treatments (16.87 kJ/L) than in millisecond treatments (150 kJ/L). In terms of the specific energy required to cause microalgae inactivation, treatments in the microsecond range also resulted in greater energy efficiency. The comparison of extraction yields in the range of milliseconds (5 kV, 20 ms) and microseconds (20, 25 pulses) under the conditions in which the maximum extraction was observed revealed that the improvement in the carotenoid extraction was similar and chlorophyll a and b extraction was slightly higher for treatments in the microsecond range. The specific energy that was required for the treatment in the millisecond range (150 kJ/L) was much higher than those required in the microsecond range (30 kJ/L). The comparison of the efficacy of both types of pulses on the extraction enhancement just after the treatment and after a post-pulse incubation period seemed to indicate that PEF in the millisecond range created irreversible alterations while, in the microsecond range, the defects were a dynamic structure along the post-pulse time that caused a subsequent increment in the extraction yield.

E. coli electroeradication on a closed loop circuit by using milli-, micro- and nanosecond pulsed electric fields: comparison between energy costs.

One of the different ways to eradicate microorganisms, and particularly bacteria that might have an impact on health consists in the delivery of pulsed electric fields (PEFs). The technologies of millisecond (ms) or microsecond (µs) PEF are still well known and used for instance in the process of fruit juice sterilization. However, this concept is costly in terms of delivered energy which might be too expensive for some other industrial processes. Nanosecond pulsed electric fields (nsPEFs) might be an alternative at least for lower energetic cost. However, only few insights were available and stipulate a gain in cost and in efficiency as well. Using Escherichia coli, the impact of frequency and low rate on eradication and energy consumption by msPEF, µsPEF and nsPEF have been studied and compared. While a 1 log10 was reached with an energy cost of 100 and 158 kJ/L with micro- and millisecond PEFs respectively, nsPEF reached the reduction for similar energy consumption. The best condition was obtained for a 1 log10 deactivation in 0.5h, for energy consumption of 143 kJ/L corresponding to 0.04 W · h when the field was around 100 kV/cm. Improvement can also be expected by producing a generator capable to increase the electric field.

Optimization of protein electroextraction from microalgae by a flow process.

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 µm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size.

Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome.

A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)-mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII), oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant) and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin) reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII-mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism.