Evaluation of the neurotoxic effects of engineered nanomaterials in C57BL/6J mice in 28-day oral exposure studies.

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays. Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.

Surface-engineered nanoliposomes with lipidated and non-lipidated peptide-dendrimeric scaffold for efficient transdermal delivery of a therapeutic agent: Development, characterization, toxicological and preclinical performance analyses.

The current study aimed to develop novel peptide dendrimer (PD)-conjugated nanoliposomal formulations of asenapine maleate (ASP) for improvement in the transdermal delivery and pharmacokinetic profile of the drug. Novel arginine-terminated PDs (+/-lipidation) were prepared by solid phase peptide synthesis, followed by conjugation onto ASP nanoliposomes. The nanoliposomes were characterized for particle size (and polydispersity index), zeta potential (ZP), drug entrapment efficiency, shape and morphology, differential scanning calorimetry and FT-IR spectroscopy. Ex vivo skin permeation and retention studies demonstrated considerably higher percutaneous permeation of ASP from the developed nanoliposomes (Q24 = 794.31 ± 54.89 µg/cm2, Jss = 105.40 ± 4.8 µg/cm2/h, ER = 36.85 ± 2.89 for liposomes with lipidated peptide dendrimer (Lipo-PD2)) in comparison with passive diffusion studies (Q24 = 63.09 ± 3.56 µg/cm2, Jss = 3.01 ± 0.23 µg/cm2/h). Confocal Laser Scanning Microscopy (CLSM) confirmed the higher percutaneous penetration of Lipo-PD2 in comparison with liposomes without the dendrimer. In vitro cytotoxicity determined on HaCaT cell line demonstrated CTC50 of >1000 µg/mL for both the synthesized PDs and Lipo-PD2. Pharmacokinetic studies in male Sprague Dawley rats revealed considerably and significantly higher t1/2 = 82.32 ± 14.48 h and AUC0-t = 4403.34 ± 367.10 h.ng/mL, from the developed formulation, compared to orally administered ASP (t1/2 = 21.64 ± 2.53 h and AUC0-t = 2303.55 ± 444.5 h.ng/mL), demonstrating higher bioavailability and longer retention in vivo. Additionally, in vivo skin retention, brain uptake studies and pharmacodynamics of the developed formulations were investigated. Stability studies indicated that the formulations were stable up to relatively stable with respect to size, ZP and drug content for 4 months at the tested conditions. This study demonstrates that the developed PD-conjugated nanoliposomal formulations can effectively serve as a transdermal delivery strategy for ASP.

A general strategy for diversifying complex natural products to polycyclic scaffolds with medium-sized rings.

The interrogation of complex biological pathways demands diverse small molecule tool compounds, which can often lead to important therapeutics for the treatment of human diseases. Since natural products are the most valuable source for the discovery of therapeutics, the derivatization of natural products has been extensively investigated to generate molecules for biological screenings. However, most previous approaches only modified a limited number of functional groups, which resulted in a limited number of skeleta. Here we show a general strategy for the preparation of a library of complex small molecules by combining state-of-the-art chemistry - the site-selective oxidation of C-H bonds - with reactions that expand rigid, small rings in polycyclic steroids to medium-sized rings. This library occupies a unique chemical space compared to selected diverse reference compounds. The diversification strategy developed herein for steroids can also be expanded to other types of natural products.

Engineered polymeric microspheres obtained by multi-step method as potential systems for transarterial embolization and intraoperative imaging of HCC: Preliminary evaluation.

The aim of this study was the development of novel fluorescent microspheres as embolic agent for transarterial embolization (TAE) of advanced stages of hepatocellular carcinoma (HCC). TAE is a minimally invasive procedure that induces tumour regression blocking the blood flow by injection of microparticles. The microspheres currently used in clinical application cannot be visualized in vivo. Surgeon could exploit the intraoperative detection of embolic agents during resection of the malignant mass. Biocompatible indocyanine green (ICG)-loaded microspheres (CAB-CS-ICG) were prepared using a multi-step method. Chitosan (CS)-ICG particles were prepared via spray-dryer and then loaded into cellulose acetate butyrate (CAB) microspheres, fabricated by emulsion solvent extraction method. Technological parameters such as yield, size, encapsulation efficiency and morphology were studied. CAB-CS-ICG microspheres showed spherical shape and smooth surface, as well as good injectability through a 21 G×1½ needle. ICG release from CAB-CS-ICG was very low due to the strong interaction between CS and ICG. This result was also confirmed by in vitro fluorescence imaging studies, conducted using Photodynamic Eye (PDE) for the detection of particles incubated in human plasma. CAB-CS-ICG were capable to maintain the fluorescence selectivity for 4weeks. Our data suggested the potential usefulness of CAB-CS-ICG in TAE application as embolic agents and following imaging of tumour during surgical procedure.

Application of spray-drying and electrospraying/electospinning for poorly water-soluble drugs: a particle engineering approach.

Solid dispersions have been widely studied as an attractive formulation strategy for the increasingly prevalent poorly water-soluble drug compounds, including herbal medicines, often leading to improvements in drug dissolution rate and bioavailability. However, several challenges are encountered with solid dispersions, for instance regarding their physical stability, and the full potential of these formulations has yet to be reached. Solid dispersions have mainly been used to produce immediate release systems using water-soluble polymers but an extended release system may provide equal or better performance due to enhancement in the pharmacokinetics and low variability in plasma concentration. Progress in processing technologies and particle engineering provides new opportunities to prepare particle-based solid dispersions with control of physical characteristics and tailored drug release kinetics. Spray-drying and electrospraying are both technologies that allow production and continuous manufacturing of particle-based amorphous solid dispersions in a single step process and electrospinning further allows the production of fiber based systems. This review presents the use of spray drying and electrospraying/electrospinning as techniques for preparing particle-based solid dispersions, describes the particle formation processes via numerical and experimental models and discusses particle engineering using these techniques. Examples are given on the applications of these techniques for preparing solid dispersions and the challenges associated with the techniques such as stability, preparation of final dosage form and scale-up are also discussed.

Chemically engineered extracts: source of bioactive compounds.

Biological research and drug discovery critically depend on access to libraries of small molecules that have an affinity for biomacromolecules. By virtue of their sustained success as sources of lead compounds, natural products are recognized as "privileged" starting points in structural space for library development. Compared with synthetic compounds, natural products have distinguishing structural properties; indeed, researchers have begun to quantify and catalog the differences between the two classes of molecules. Measurable differences in the number of chiral centers, the degree of saturation, the presence of aromatic rings, and the number of the various heteroatoms are among the chief distinctions between natural and synthetic compounds. Natural products also include a significant proportion of recurring molecular scaffolds that are not present in currently marketed drugs: the bioactivity of these natural substructures has been refined over the long process of evolution. In this Account, we present our research aimed at preparing libraries of semisynthetic compounds, or chemically engineered extracts (CEEs), through chemical diversification of natural products mixtures. The approach relies on the power of numbers, that is, in the chemical alteration of a sizable fraction of the starting complex mixture. Major changes in composition can be achieved through the chemical transformation of reactive molecular fragments that are found in most natural products. If such fragments are common enough, their transformation represents an entry point for chemically altering a high proportion of the components of crude natural extracts. We have searched for common reactive fragments in the Dictionary of Natural Products (CRC Press) and identified several functional groups that are expected to be present in a large fraction of the components of an average natural crude extract. To date, we have used reactions that incorporate (i) nitrogen atoms through carbonyl groups, (ii) sulfur by transformation of -OH and amines, and (iii) bromine through double bonds and aromatic rings. The resulting CEEs had different composition and biomolecular properties than their natural progenitors. We isolated a semisynthetic ß-glucosidase inhibitor from a CEE prepared by reaction with benzenesulfonyl chloride, an antifungal pyrazole from a CEE prepared by reaction with hydrazine, and an acetylcholinesterase inhibitor from a CEE prepared through bromination. Our results illustrate how biological activity can be generated through chemical diversification of natural product mixtures. Moreover, the level of control that can be asserted in the process by judicious design and experimental choices underscores the potential for further development of CEEs in both basic research and drug discovery.

Application of screening methods, shape signatures and engineered biosensors in early drug discovery process.

PURPOSE: In this study, two unreported estrogen antagonists were identified using a combination of computational screening and a simple bacterial estrogen sensor. METHODS: Molecules here presented were initially part of a group obtained from a library of over a half million chemical compounds, using the Shape Signatures method. The structures within this group were then clustered and compared to known antagonists based on their physico-chemical parameters, and possible binding modes of the compounds to the Estrogen Receptor alpha (ER alpha) were analyzed. Finally, thirteen candidate compounds were purchased, and two of them were shown to behave as potential subtype-selective estrogen antagonists using a set of bacterial estrogen biosensors, which included sensors for ER alpha, ER beta, and a negative control thyroid hormone beta biosensor. These activities were then analyzed using an ELISA assay against activated ER alpha in human MCF-7 cell extract. RESULTS: Two new estrogen receptor antagonists were detected using in silico Shape Signatures method with an engineered subtype-selective bacterial estrogen biosensor and commercially available ELISA assay. Additional thyroid biosensor control experiments confirmed no compounds interacted with human thyroid receptor beta. CONCLUSIONS: This work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.

Triple hydrogen bonds direct crystal engineering of metal-assembled complexes: the effect of a novel organic-inorganic module on supramolecular structure.

Novel triply hydrogen bonded suprastructures based on [M(tdpd)2(L)2]2- (H2tdpd=1,4,5,6-tetrahydro-5,6-dioxo-2,3-pyrazinedicarbonitrile, L=solvent) and melamine-analogous cations have been synthesized and characterized. The use of anions containing two AAA sets from [M(tdpd)2(L)2]2- together with cations containing one DDD set (A=hydrogen-bond acceptor, D=hydrogen-bond donor) leads to the formation of complementary triply hydrogen bonded modules in the solid state. In all cases, the building module is further extended via additional hydrogen-bonding interactions to produce a tape, and tapes are assembled into sheets. These results show that a hydrogen-bonded module consisting of different kinds of building blocks, one of which is a metal complex that includes hydrogen-bond acceptor sites and the other is a hydrogen-bond donor molecule, will be attractive for constructing metal-containing supramolecular systems by the self-assembly technique.

Artificial metalloenzymes based on biotin-avidin technology for the enantioselective reduction of ketones by transfer hydrogenation.

Most physiological and biotechnological processes rely on molecular recognition between chiral (handed) molecules. Manmade homogeneous catalysts and enzymes offer complementary means for producing enantiopure (single-handed) compounds. As the subtle details that govern chiral discrimination are difficult to predict, improving the performance of such catalysts often relies on trial-and-error procedures. Homogeneous catalysts are optimized by chemical modification of the chiral environment around the metal center. Enzymes can be improved by modification of gene encoding the protein. Incorporation of a biotinylated organometallic catalyst into a host protein (avidin or streptavidin) affords versatile artificial metalloenzymes for the reduction of ketones by transfer hydrogenation. The boric acid.formate mixture was identified as a hydrogen source compatible with these artificial metalloenzymes. A combined chemo-genetic procedure allows us to optimize the activity and selectivity of these hybrid catalysts: up to 94% (R) enantiomeric excess for the reduction of p-methylacetophenone. These artificial metalloenzymes display features reminiscent of both homogeneous catalysts and enzymes.

Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis.

Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3- to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyltetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.

Full-speed mammalian genetics: in vivo target validation in the drug discovery process.

The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.