Grafting TRAIL through Either Amino or Carboxylic Groups onto Maghemite Nanoparticles: Influence on Pro-Apoptotic Efficiency.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF cytokine superfamily. TRAIL is able to induce apoptosis through engagement of its death receptors DR4 and DR5 in a wide variety of tumor cells while sparing vital normal cells. This makes it a promising agent for cancer therapy. Here, we present two different ways of covalently grafting TRAIL onto maghemite nanoparticles (NPs): (a) by using carboxylic acid groups of the protein to graft it onto maghemite NPs previously functionalized with amino groups, and (b) by using the amino functions of the protein to graft it onto NPs functionalized with carboxylic acid groups. The two resulting nanovectors, NH-TRAIL@NPs-CO and CO-TRAIL@NPs-NH, were thoroughly characterized. Biological studies performed on human breast and lung carcinoma cells (MDA-MB-231 and H1703 cell lines) established these nanovectors are potential agents for cancer therapy. The pro-apoptotic effect is somewhat greater for CO-TRAIL@NPs-NH than NH-TRAIL@NPs-CO, as evidenced by viability studies and apoptosis analysis. A computational study indicated that regardless of whether TRAIL is attached to NPs through an acid or an amino group, DR4 recognition is not affected in either case.

TRAIL acts synergistically with iron oxide nanocluster-mediated magneto- and photothermia.

Targeting TRAIL (Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand) receptors for cancer therapy remains challenging due to tumor cell resistance and poor preparations of TRAIL or its derivatives. Herein, to optimize its therapeutic use, TRAIL was grafted onto iron oxide nanoclusters (NCs) with the aim of increasing its pro-apoptotic potential through nanoparticle-mediated magnetic hyperthermia (MHT) or photothermia (PT). Methods: The nanovector, NC@TRAIL, was characterized in terms of size, grafting efficiency, and potential for MHT and PT. The therapeutic function was assessed on a TRAIL-resistant breast cancer cell line, MDA-MB-231, wild type (WT) or TRAIL-receptor-deficient (DKO), by combining complementary methylene blue assay and flow cytometry detection of apoptosis and necrosis. Results: Combined with MHT or PT under conditions of "moderate hyperthermia" at low concentrations, NC@TRAIL acts synergistically with the TRAIL receptor to increase the cell death rate beyond what can be explained by the mere global elevation of temperature. In contrast, all results are consistent with the idea that there are hotspots, close to the nanovector and, therefore, to the membrane receptor, which cause disruption of the cell membrane. Furthermore, nanovectors targeting other membrane receptors, unrelated to the TNF superfamily, were also found to cause tumor cell damage upon PT. Indeed, functionalization of NCs by transferrin (NC@Tf) or human serum albumin (NC@HSA) induces tumor cell killing when combined with PT, albeit less efficiently than NC@TRAIL. Conclusions: Given that magnetic nanoparticles can easily be functionalized with molecules or proteins recognizing membrane receptors, these results should pave the way to original remote-controlled antitumoral targeted thermal therapies.

The encapsulation of the gemcitabine anticancer drug into grapheme nest: a theoretical study.

The efficient transport of a drug molecule until its target cell constitutes a significant challenge for delivery processes. To achieve such objectives, solid nanocapsules that protect the immune system during the transport should be developed and controlled at the nanoscale level. From this point of view, nanostructures based on graphene sheets could present some promising properties due to their ultimate size and dimension. In this work, we present theoretical results using DFT calculations, dealing with a graphene-based delivery system. Indeed, we demonstrate the stability of the gemcitabine anticancer molecule when it is encapsulated into two concave graphene sheets organized as a nest. Quantum calculations showed that the most stable state is located inside the nest, which is then formed by two layers distanced 6 Å from each other. For all the optimized systems, we focused on the dependence of the interaction energy on the molecule displacements during its entrance in the graphene nest and its exit from it. We also analyzed their consequence on the local morphological and electronic charge properties. Graphical Abstract Adsorption energy (in eV) of gemcitabine drug during its encapsulation inside the nest of grapheneand its release from it.

Improved photodynamic effect through encapsulation of two photosensitizers in lipid nanocapsules.

Photodynamic therapy (PDT) has developed into a new clinical and non-invasive treatment for cancer over the past 30 years. By the combination of three non-toxic partners, i.e. a photosensitizer (PS), molecular oxygen (O2) and light, cytotoxic reactive oxygen species (ROS) are locally produced leading to irreversible vascular and cellular damage. In the present study, we report for the first time that the combination of two photosensitizers (2 PSs: Protoporphyrin IX, PpIX and Hypericin, Hy) loaded in the same lipid nanocapsules (LNCs) leads to enhanced photodynamic therapy efficiency when compared with previously reported systems. The 2 PS-loaded LNCs are shown to increase the in vitro phototoxicity at the nanomolar range (IC50 = 274 and 278 nM on HeLa and MDA-MB-231 cell lines, respectively), whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Intracellular localization in HeLa cells indicates a subcellular asymmetry of PpIX and Hy, in the plasma, ER membranes and round internal structures. The biodistribution of LNCs was studied upon different routes of injection into Swiss nude mice; based on the obtained data, LNCs were injected intratumorally and used to slow the growth of xenograft tumors in mice. The results obtained in this study suggest that the combination of two or more PSs may be a promising strategy to improve the efficacy of conventional photodynamic therapy as well as to reduce dark toxicity.

Controlled modification of electrochemical microsystems with polyethylenimine/reduced graphene oxide using electrophoretic deposition: Sensing of dopamine levels in meat samples.

Microsystems play an important role in many biological and environmental applications. The integration of electrical interfaces into such miniaturized systems provides new opportunities for electrochemical sensing where high sensitivity and selectivity towards the analyte are requested. This can be only achieved upon controlled functionalization of the working electrode, a challenge for compact microsystems. In this work, we demonstrate the benefit of electrophoretic deposition (EPD) of reduced graphene oxide/polyethylenimine (rGO/PEI) for the selective modification of a gold (Au) microelectrode in a microsystem comprising a Pt counter and a Ag/AgCl reference electrode. The functionalized microsystem was successfully applied for the sensing of dopamine with a detection limit of 50nM. Additionally, the microsystem exhibited good performance for the detection of dopamine levels in meat samples.

Controlling activation barrier by carbon nanotubes as nano-chemical reactors.

The oxidative addition of primary amine on a monocyclic phospholane was studied in confined conditions. This one-step chemical reaction has been investigated using the DFT technique to elucidate the role of confinement in carbon nanotubes on the reaction. Calculations were carried out by a progressive increase of the nanotube diameters from 10 Å to 15 Å in order to highlight the dependence of the reactivity on the nanotube diameter. First, single point investigations were dedicated to the study of reactants, transition states, and products placed in the different nanotubes while keeping their optimized structure as free compounds. Second, all studied compounds were relaxed inside nanotubes and their geometries were fully optimized. Within these approaches, we proved that the activation barrier could be controlled depending on the confinement, generating a well-controlled catalysis process.

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

Carbon nanotubes as gene carriers: Focus on internalization pathways related to functionalization and properties.

Carbon nanotubes represent promising transporters for delivery of DNA and other biomolecules into living cells. Various methods of CNTs surface functionalization have been developed. These are essential to improve CNTs dispersibility and permit their interactions with biological structures that broaden their use in advanced biomedical applications. The present review discusses the different single walled carbon nanotubes and multiwalled carbon nanotubes functionalization methods, leading to the formation of optimized and functionalized-CNT complexes with DNA. F-CNTs are recognized as efficient and promising gene carriers. Emphasis is then placed on the processes used by f-CNTs/DNA complexes to cross cell membranes. Energy independent pathways and uptake mechanisms dependent on energy, such as endocytosis or phagocytosis, are reported by many studies, and if these mechanisms seem contradictory at first sight, a detailed review of the literature illustrates that they are rather complementary. Preferential use of one or the other depends on the DNA and CNTs chemical nature and physical parameters, experimental procedures and cell types. STATEMENT OF SIGNIFICANCE: Efficient non-viral gene delivery is desirable, yet challenging. CNTs appear as a promising solution to penetrate into cells and successfully deliver DNA. Moreover, the field of use of CNTs as gene carrier is large and is currently growing. This critical review summarizes the development and evaluation of CNTs as intracellular gene delivery system and provides an overview of functionalized CNTs/DNA cellular uptake mechanisms, depending on several parameters of CNTs/DNA complexes.

Biomimetic solution against dewetting in a highly hydrophobic nanopore.

A water molecule is the foundation of life and is the primary compound in every living system. While many of its properties are understood in a bulk solvent, its behavior in a small hydrophobic nanopore still raises fundamental questions. For instance, a wetting/dewetting transition in a hydrophobic solid-state or a polymer nanopore occurs stochastically and can only be prevented by external physical stimuli. Controlling these transitions would be a primary requirement to improve many applications. Some biological channels, such as gramicidin A (gA) proteins, show a high rate of water and ion diffusion in their central subnanochannel while their external surface is highly hydrophobic. The diameter of this channel is significantly smaller than the inner size of the lowest artificial nanopore in which water drying occurs (i.e. 1.4 nm). In this paper, we propose an innovative idea to generate nanopore wetting as a result of which the application of an external field is no longer required. In a nanopore, the drying or wetting of the inner walls occurs randomly (in experiments and in simulations). However, we have shown how the confinement of gA, in a dried hydrophobic nanopore, rapidly generates a stable wetting of the latter. We believe that this simple idea, based on biomimetism, could represent a real breakthrough that could help to improve and develop new nanoscale applications.

Nanovectorization of DNA Through Cells Using Protamine Complexation.

Carbon nanotubes (CNT) are currently used as a promising family of nanovectors able to deliver different types of therapeutic molecules. Several applications dealing with CNT used as drug nanocarriers have been developed since their ability to penetrate into the cells has been proved. CNT can thus load several active molecules to various cells. In this paper, we will use molecular dynamic simulation to describe theoretically the potential of CNT to transport and deliver DNA through the formation of protamine-DNA-CNT complex.

Enhanced DR5 binding capacity of nanovectorized TRAIL compared to its cytotoxic version by affinity chromatography and molecular docking studies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of cancer cells when bound to its cognate receptors, TRAIL-R1 and TRAIL-R2 (DR4 and DR5), without being toxic to healthy cells. Nanovectorized TRAIL (abbreviated as NPT) is 10 to 20 times more efficient than one of the most potent soluble TRAIL used in preclinical studies (His-TRAIL). To determine whether differences in affinity may account for NPT superiority, a thermodynamic study was undertaken to evaluate NPT versus TRAIL binding affinity to DR5. Docking calculations showed that TRAIL in homotrimer configuration was more stable than in heterotrimer, because of the presence of one Zn ion in its structure. Indeed, TRAIL trimers can have head-to-tail orientations when Zn is missing. Altogether these data suggest that TRAIL homotrimer structures are predominant in solution and then are grafted on NPT. When docked to DR5, NPT carrying TRAIL homotrimer leads to a more stable complex than TRAIL monomer-based NPT. To comfort these observations, the extracellular domain of DR5 was immobilized on a chromatographic support using an "in situ" immobilization technique. The determination of the thermodynamic data (enthalpy ∆H° and entropy ∆S°*) of TRAIL and NPT binding to DR5 showed that the binding mechanism was pH dependent. The affinity of NPT to DR5 increased with pH, and the ionized energy was more important for NPT than for soluble TRAIL. Moreover, because of negative values of ∆H° and ∆S°* quantities, we demonstrated that van der Waals and hydrogen bonds governed the strong NPT-DR5 association for pH > 7.4 (as for TRAIL alone). Copyright © 2016 John Wiley & Sons, Ltd.

Pulse Width Modulation Applied to Olfactory Stimulation for Intensity Tuning.

For most olfactometers described in the literature, adjusting olfactory stimulation intensity involves modifying the dilution of the odorant in a neutral solution (water, mineral, oil, etc.), the dilution of the odorant air in neutral airflow, or the surface of the odorant in contact with airflow. But, for most of these above-mentioned devices, manual intervention is necessary for adjusting concentration. We present in this article a method of controlling odorant concentration via a computer which can be implemented on even the most dynamic olfactometers. We used Pulse Width Modulation (PWM), a technique commonly used in electronic or electrical engineering, and we have applied it to odor delivery. PWM, when applied to odor delivery, comprises an alternative presentation of odorant air and clean air at a high frequency. The cycle period (odor presentation and rest) is 200 ms. In order to modify odorant concentration, the ratio between the odorant period and clean air presentation during a cycle is modified. This ratio is named duty cycle. Gas chromatography measurements show that this method offers a range of mixing factors from 33% to 100% (continuous presentation of odor). Proof of principle is provided via a psychophysical experiment. Three odors (isoamyl acetate, butanol and pyridine) were presented to twenty subjects. Each odor was delivered three times with five values of duty cycles. After each stimulation, the subjects were asked to estimate the intensity of the stimulus on a 10 point scale, ranging from 0 (undetectable) to 9 (very strong). Results show a main effect of the duty cycles on the intensity ratings for all tested odors.

Theoretical demonstration of the potentiality of boron nitride nanotubes to encapsulate anticancer molecule.

Anticancer drug transport is now becoming an important scientific challenge since it would allow localizing the drug release near the tumor cell, avoiding secondary medical effects. We present theoretical results, based on density functional theory and molecular dynamics simulations, which demonstrate the stability of functionalized single (10,10) boron nitride nanotubes (BNNTs) filled with anticancer molecule such as carboplatin (CPT). For this functionalized system we determine the dependence of the adsorption energy on the molecule displacement near the inner BNNTs surface, together with their local morphological and electrical changes and compare the values to the adsorption energy obtained on the outer surface. Quantum simulations show that the most stable physisorption state is located inside the nanotube, with no net charge transfer. This demonstrates that chemotherapeutic encapsulation is the most favorable way to transport drug molecules. The solvent effect and dispersion repulsion contributions are then taken into account using molecular dynamics simulations. Our results confirm that carboplatin therapeutic agents are not affected when they are adsorbed inside BNNTs by the surrounding water molecules.

Donepezil-ferulic acid hybrids as anti-Alzheimer drugs.

BACKGROUND: Due to the complex nature of Alzheimer's disease, there is a renewed and growing search for multitarget drugs. RESULTS: Donepezil-ferulic acid hybrids (DFAHs) were prepared by the one-pot Ugi-4CR in low-to-moderate yields. DFAHs are potent antioxidant agents, showing oxygen radical absorbance capacity values in the range 4.80-8.71 trolox equivalents, quite higher compared with those recorded for ferulic acid and melatonin. From the ChEs inhibition studies, we conclude that DFAH 8, bearing an ethylene linker, and DFAH 12, bearing a propylene linker, both substituted with a melatonin motif, are the most potent inhibitors, in the nanomolar range. CONCLUSION: We have identified DFAH 8 as a very potent antioxidant, and totally selective equineButyrylCholinEsterase (eqBuChE) inhibitor.

Nanovectorization of TRAIL with single wall carbon nanotubes enhances tumor cell killing.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor (TNF) superfamily. This type II transmembrane protein is able to bound specifically to cancer cell receptors (i.e., TRAIL-R1 (or DR4) and TRAIL-R2 (or DR5)) and to induce apoptosis without being toxic for healthy cells. Because membrane-bound TRAIL induces stronger receptor aggregation and apoptosis than soluble TRAIL, we proposed here to vectorize TRAIL using single-walled carbon nanotubes (SWCNTs) to mimic membrane TRAIL. Owing to their exceptional and revolutional properties, carbon nanotubes, especially SWCNTs, are used in a wide range of physical or, now, medical applications. Indeed due to their high mechanical resistance, their high flexibility and their hydrophobicity, SWCNTs are known to rapidly diffuse in an aqueous medium such as blood, opening the way of development of new drug nanovectors (or nanocarriers). Our TRAIL-based SWCNTs nanovectors proved to be more efficient than TRAIL alone death receptors in triggering cancer cell killing. These NPTs increased TRAIL pro-apoptotic potential by nearly 20-fold in different Human tumor cell lines including colorectal, nonsmall cell lung cancer, or hepatocarcinomas. We provide thus a proof-of-concept that TRAIL nanovector derivatives based on SWCNT may be useful to future nanomedicine therapies.

Encapsulation into carbon nanotubes and release of anticancer Cisplatin drug molecule.

Molecular dynamics simulations have been investigated to study the interactions between single-wall carbon nanotubes and an anticancer agent Pt complex (Cisplatin). The optimized diameter of the vector system has been determined to encapsulate in the best conditions the drug molecules. The simulation results show also that several drug molecules can be adsorbed inside the nanotubes, leading to an increased confinement time. Moreover, our simulations show that the release of the drug near a cell membrane model is favored, opening the way to a natural drug nanocapsule.

Novel tacrine-grafted Ugi adducts as multipotent anti-Alzheimer drugs: a synthetic renewal in tacrine-ferulic acid hybrids.

Herein we describe the design, multicomponent synthesis, and biological, molecular modeling and ADMET studies, as well as in vitro PAMPA-blood-brain barrier (BBB) analysis of new tacrine-ferulic acid hybrids (TFAHs). We identified (E)-3-(hydroxy-3-methoxyphenyl)-N-{8[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-N-[2-(naphthalen-2-ylamino)2-oxoethyl]acrylamide (TFAH 10 n) as a particularly interesting multipotent compound that shows moderate and completely selective inhibition of human butyrylcholinesterase (IC50 =68.2 nM), strong antioxidant activity (4.29 equiv trolox in an oxygen radical absorbance capacity (ORAC) assay), and good ß-amyloid (Aß) anti-aggregation properties (65.6 % at 1:1 ratio); moreover, it is able to permeate central nervous system (CNS) tissues, as determined by PAMPA-BBB assay. Notably, even when tested at very high concentrations, TFAH 10 n easily surpasses the other TFAHs in hepatotoxicity profiling (59.4 % cell viability at 1000 µM), affording good neuroprotection against toxic insults such as Aß1-40 , Aß1-42 , H2 O2 , and oligomycin A/rotenone on SH-SY5Y cells, at 1 µM. The results reported herein support the development of new multipotent TFAH derivatives as potential drugs for the treatment of Alzheimer's disease.

Quantum study of boron nitride nanotubes functionalized with anticancer molecules.

Full DFT-D2 calculations were carried out to study the interactions between single wall (10,10) boron nitride nanotubes (BNNTs) and different molecules, such as azomethine (C2H5N) and an anticancer agent (Pt(IV) complex) linked to an amino-derivative chain. The geometry of the (10,10) BNNT-azomethine and the BNNT-amino derivative system was optimised by considering different molecular configurations on the inner and outer surfaces of the nanotube. Simulation results showed that the most stable physisorption state for both molecules was located inside the nanotube in a parallel configuration. We showed also that the molecular chemisorption was possible only when the azomethine was present above two adjacent B and N atoms of a hexagon. The attachment of an azomethine plus a subsequent drug did not perturb the cycloaddition process. Moreover, all theoretical results showed that the therapeutic agent complex was not affected when it was attached onto BNNTs.

Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and drug co-delivery and cell imaging.

The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 ± 3.9 nm) with monodisperse size characteristics (PI < 0.2) and positive surface charge up to +18.7 mV. The PEI-modified LNCs (LNC25-T) displayed good buffering capacity. Moreover, the cationic LNC25-T were able to condense DNA and form complexes via electrostatic interactions in a typical weight ratio-dependent relationship. It was found that the mean diameter of LNC25-T/pDNA complexes increased to ∼40-50 nm with the LNC25-T/pDNA ratio from 1 to 500. Gel electrophoresis and cell viability experiments showed that the LNC25-T/pDNA complexes had high stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.