In vivo observation of gold nanoparticles in the central nervous system of <Emphasis Type="Italic">Blaberus discoidalis</Emphasis>

Background  

Nanoparticles (NPs) are widely studied for biomedical applications. Understanding interactions between NPs and biomolecules or cells has yet to be achieved. Here we present a novel in vivo method to study interactions between NPs and the nervous system of the discoid or false dead-head roach, Blaberus discoidalis. The aims of this study were to present a new and effective method to observe NPs in vivo that opens the door to new methods of study to observe the interactions between NPs and biological systems and to present an inexpensive and easy-to-handle biological system.     

Acute Toxicity of Intravenously Administered Titanium Dioxide Nanoparticles in Mice

Background

With a wide range of applications, titanium dioxide (TiO₂) nanoparticles (NPs) are manufactured worldwide in large quantities. Recently, in the field of nanomedicine, intravenous injection of TiO₂ nanoparticulate carriers directly into the bloodstream has raised public concerns on their toxicity to humans.

Methods

In this study, mice were injected intravenously with a single dose of TiO₂ NPs at varying dose levels (0, 140, 300, 645, or 1387 mg/kg). Animal mortality, blood biochemistry, hematology, genotoxicity and histopathology were investigated 14 days after treatment.

Results

Death of mice in the highest dose (1387 mg/kg) group was observed at day two after TiO₂ NPs injection. At day 7, acute toxicity symptoms, such as decreased physical activity and decreased intake of food and water, were observed in the highest dose group. Hematological analysis and the micronucleus test showed no significant acute hematological or genetic toxicity except an increase in the white blood cell (WBC) count among mice 645 mg/kg dose group. However, the spleen of the mice showed significantly higher tissue weight/body weight (BW) coefficients, and lower liver and kidney coefficients in the TiO₂ NPs treated mice compared to control. The biochemical parameters and histological tissue sections indicated that TiO₂ NPs treatment could induce different degrees of damage in the brain, lung, spleen, liver and kidneys. However, no pathological effects were observed in the heart in TiO₂ NPs treated mice.

Conclusions

Intravenous injection of TiO₂ NPs at high doses in mice could cause acute toxicity effects in the brain, lung, spleen, liver, and kidney. No significant hematological or genetic toxicity was observed.     

ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels

Background

The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate.

Results

The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO₂ NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels.

Conclusion

We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.

     

Biodistribution and Trafficking of Hydrogel Nanoparticles in Adult Mosquitoes

Background

Nanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes.

Methodology/Principal Findings

PRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract.

Conclusions/Significance

Herein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.     

Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

Background  

Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features.     

Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids

Background

Protein Kinases are key regulators of cell function and play essential roles in the occurrence and development of many human diseases. Many kinase inhibitors have been used for molecular targeted treatment of those diseases such as cancer and inflammation. However, those highly hydrophobic kinase inhibitors shared the common features of poor bioavailability and limited in vivo half-life, which strongly impeded their practical applications. Our previous study demonstrated that microbial synthesized biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a member of polyhydroxyalkanoates (PHAs) family, could serve as a promising delivery nanocarrier for those hydrophobic kinase inhibitors. Recently, a novel natural synthesized hybrid copolymer, PEG200 end-capped PHBHHx (PHBHHxPEG) was produced by Aeromonas hydrophila fermentation. In this study, the novel PHBHHxPEG NPs were prepared and investigated to serve as intracellular delivery nanocarriers for sustained release of hydrophobic kinase inhibitors.

Results

PHBHHxPEG nanoparticles (NPs) prepared by an emulsification–solvent evaporation method were spherical with a diameter around 200 nm. The entrapment efficiency on rapamycin in PHBHHxPEG NPs was 91.9% and the sustained release of rapamycin from PHBHHxPEG NPs could be achieved for almost 10 days. The cellular uptake of PHBHHxPEG NPs was significant higher than that of PHBHHx NPs. The anti-proliferation effect and mTOR inhibition ability of rapamycin-loaded PHBHHxPEG NPs was stronger than that of drug-loaded PHBHHx NPs and free rapamycin.

Conclusions

PHBHHxPEG NPs could achieve the efficient entrapment and sustained release of rapamycin. The novel biodegradable PHBHHxPEG appeared a promising nanocarrier for sustained delivery of hydrophobic kinase inhibitors with improved cellular uptake and kinase inhibition efficiency.     

Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications

Background  

EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions.     

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles

Background

Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known.

Antimicrobial effects of zero-valent iron nanoparticles on gram-positive <Emphasis Type="Italic">Bacillus</Emphasis> strains and gram-negative <Emphasis Type="Italic">Escherichia coli</Emphasis> strains

Background

Zero-valent iron nanoparticles (ZVI NPs) have been used extensively for the remediation of contaminated soil and groundwater. Owing to their large active surface area, they serve as strong and effective reductants. However, the ecotoxicity and bioavailability of ZVI NPs in diverse ecological media have not been evaluated in detail and most studies have focused on non-nano ZVI or Fe⁰. In addition, the antimicrobial properties of ZVI NPs have rarely been investigated, and the underlying mechanism of their toxicity remains unknown.

Results

In the present study, we demonstrate that ZVI NPs exhibited significant toxicity at 1000 ppm against two distinct gram-positive bacterial strains (Bacillus subtilis 3610 and Bacillus thuringiensis 407) but not against two gram-negative strains (Escherichia coli K12 and ATCC11634). Specifically, ZVI NPs caused at least a 4-log and 1-log reductions in cell numbers, respectively, in the two Bacillus strains, whereas no change was detected in the two E. coli strains. X-ray photoelectron spectroscopy, X-ray absorption near-edge, and extended X-ray absorption fine structure spectra confirmed that Bacillus cells exposed to ZVI NPs contained mostly Fe₂O₃ with some detectable FeS. This finding indicated that Fe⁰ nanoparticles penetrated the bacterial cells, where they were subsequently oxidized to Fe₂O₃ and FeS. RedoxSensor analysis and propidium iodide (PI) staining showed decreased reductase activity and increased PI in both Bacillus strains treated with a high (1000 ppm) concentration of ZVI NPs.

Conclusion

Taken together, these data show that the toxicity of ZVI NPs was derived from their oxidative properties, which may increase the levels of reactive oxygen species and lead to cell death.

     

Desulfurization with <Emphasis Type="Italic">Thialkalivibrio versutus</Emphasis> immobilized on magnetic nanoparticles modified with 3-aminopropyltriethoxysilane

Objective

Thialkalivibrio versutus D301 cells were immobilized on Fe₃O₄ nanoparticles (NPs) synthesized by an improved chemical coprecipitation method and modified with 3-aminopropyltriethoxysilane (APTES), then the immobilized cells were used in sulfur oxidation.

Results

The prepared Fe₃O₄–APTES NPs had a narrow size distribution (10 ± 2 nm) and were superparamagnetic, with a saturation magnetization of 60.69 emu/g. Immobilized cells had a saturation magnetization of 34.95 emu/g and retained superparamagnetism. The optimum conditions for cell immobilization were obtained at pH 9.5 and 1 M Na⁺. The immobilization capacity of Fe₃O₄–APTES NPs was 7.15 g DCW/g-NPs that was 2.3-fold higher than that of Fe₃O₄ NPs. The desulfurization efficiency of the immobilized cells was close to 100%, having the same sulfur oxidation capacity as free cells. Further, the immobilized cells could be reused at least eight times, retaining more than 85% of their desulfurization efficiency.

Conclusion

Immobilization of cells with the modified magnetic NPs efficiently increased cell controllability, have no effect on their desulfurization activity and could be effectively used in large-scale industrial applications.

     

Collagen concentration and biomechanical properties of samples from the lower uterine cervix in relation to age and parity in non-pregnant women

Background  

During normal pregnancy the cervix has a load bearing function. The cervical tissue consists mainly of an extracellular matrix (ECM) rich in collagen; important for the biomechanical properties. The aim of the present study was to evaluate how the biomechanical strength of samples from the distal cervix is associated with collagen content in relation to age and parity. This study demonstrates a method to investigate cervical tissue from women who still have their uterus in situ.     

Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis

Background

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process.

Methods

We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease.

Results

Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node–derived T cells in response to nonspecific polyclonal stimuli.

Conclusions

The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS.     

Preparation and characterization of magnetic gold nanoparticles to be used as doxorubicin nanocarriers

Magnetic targeted drug delivery (MTD), using magnetic gold nanoparticles (Fe₃O₄@Au NPs) conjugated with an anti-cancer drug is a promise modality for cancer treatment. In this study, Fe₃O₄@Au NPs were prepared and functionalized with thiol-terminated polyethylene glycol (PEG), then loaded with anti-cancer drug doxorubicin (DOX). The physical properties of the prepared NPs were characterized using different techniques. Transmission electron microscopy (TEM) revealed the mono dispersed nature of Fe₃O₄@Au NPs with an average size of 20 nm which was confirmed using Dynamic light scattering (DLS) measurements. Zeta potential measurements along with UV–VIS spectroscopy demonstrated surface DOX loading on Fe₃O₄@Au NPs. Energy Dispersive X-ray Spectroscopy (EDX) assured the existence of both iron and gold elements in the prepared NPs. The paramagnetic properties of the prepared NPs were assessed by vibrating sample magnetometer (VSM). The maximum DOX-loading capacity was 100 μg DOX/mg of Fe₃O₄@Au NPs. It was found that DOX released more readily at acidic pH. In vitro studies on MCF-7 cell line elucidated that DOX loaded Fe₃O₄@Au NPs (Fe₃O₄@Au-PEG-DOX) have more potent therapeutic effect than free DOX. Knowledge gained in this study may open the door to pursue Fe₃O₄@Au NPs as a viable nanocarriers for different molecules delivery in many diagnostic and therapeutic applications.     

Improved RNA quality and TaqMan<Superscript>®</Superscript>Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

Background  

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp).     

Safety assessment of near infrared light emitting diodes for diffuse optical measurements

Background  

Near infrared (NIR) light has been used widely to monitor important hemodynamic parameters in tissue non-invasively. Pulse oximetry, near infrared spectroscopy, and diffuse optical tomography are examples of such NIR light-based applications. These and other similar applications employ either lasers or light emitting diodes (LED) as the source of the NIR light. Although the hazards of laser sources have been addressed in regulations, the risk of LED sources in such applications is still unknown.     

Mesenchymal‐like stem cells in canine ovary show high differentiation potential

Objectives

Recent studies have reported the existence of stem cells in ovarian tissue that show enhanced proliferative and differentiation potential compared to other adult tissues. Based on this evidence, we hypothesized that ovarian tissue contained mesenchymal‐like stem cells (MSC) that could be isolated using a novel rapid plastic adhesion technique.

Materials and methods

We established MSC lines derived from ovarian and adipose tissue based on their ability to rapidly adhere to plastic culture dishes in the first 3 hours after plating and studied their potentiality in terms of molecular markers and differentiation capacity.

Results

Morphological and kinetic properties of in vitro cultured ovarian MSC were similar to adipose‐derived MSC, and both reached senescence after similar passage numbers. Ovarian‐derived MSC expressed mesenchymal (CD90 and CD44) but not haematopoietic markers (CD34 and CD45), indicating similarity to adipose‐derived MSC. Moreover, ovarian‐derived MSC expressed NANOG, TERT, SOX2, OCT4 and showed extensive capacity to differentiate not only into adipogenic, osteogenic and chondrogenic tissue but also towards neurogenic and endodermal lineages and even precursors of primordial germ cells.

Conclusion

These results show for the first time the derivation of ovarian cells with the molecular properties of MSC as well as wide differentiation potential. Canine ovarian tissue is accessible, expandable, multipotent and has high plasticity, holding promise for applications in regenerative medicine.

     

Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells

Background

Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO₂ nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO₂ Nanobyk 3810™ NPs, used in wood care, and their residues, altered by light or acid.

Results

Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs. All CeO₂ NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 μg/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810™. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.

Conclusion

The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-700) contains supplementary material, which is available to authorized users.     

Plant Extract Synthesized PLA Nanoparticles for Controlled and Sustained Release of Quercetin: A Green Approach

Background

Green synthesis of metallic nanoparticles (NPs) has been extensively carried out by using plant extracts (PEs) which have property of stabilizers/ emulsifiers. To our knowledge, there is no comprehensive study on applying a green approach using PEs for fabrication of biodegradable PLA NPs. Conventional methods rely on molecules like polyvinyl alcohol, polyethylene glycol, D-alpha-tocopheryl poly(ethylene glycol 1000) succinate as stabilizers/emulsifiers for the synthesis of such biodegradable NPs which are known to be toxic. So, there is urgent need to look for stabilizers which are biogenic and non-toxic. The present study investigated use of PEs as stabilizers/emulsifiers for the fabrication of stable PLA NPs. Synthesized PLA NPs through this green process were explored for controlled release of the well known antioxidant molecule quercetin.

Methodology/Principal Findings

Stable PLA NPs were synthesized using leaf extracts of medicinally important plants like Syzygium cumini (1), Bauhinia variegata (2), Cedrus deodara (3), Lonicera japonica (4) and Eleaocarpus sphaericus (5). Small and uniformly distributed NPs in the size range 70±30 nm to 143±36 nm were formed with these PEs. To explore such NPs for drugs/ small molecules delivery, we have successfully encapsulated quercetin a lipophilic molecule on a most uniformly distributed PLA-4 NPs synthesized using Lonicera japonica leaf extract. Quercetin loaded PLA-4 NPs were observed for slow and sustained release of quercetin molecule.

Conclusions

This green approach based on PEs mediated synthesis of stable PLA NPs pave the way for encapsulating drug/small molecules, nutraceuticals and other bioactive ingredients for safer cellular uptake, biodistribution and targeted delivery. Hence, such PEs synthesized PLA NPs would be useful to enhance the therapeutic efficacy of encapsulated small molecules/drugs. Furthermore, different types of plants can be explored for the synthesis of PLA as well as other polymeric NPs of smaller size.     

TAMEE: data management and analysis for tissue microarrays

Background  

With the introduction of tissue microarrays (TMAs) researchers can investigate gene and protein expression in tissues on a high-throughput scale. TMAs generate a wealth of data calling for extended, high level data management. Enhanced data analysis and systematic data management are required for traceability and reproducibility of experiments and provision of results in a timely and reliable fashion. Robust and scalable applications have to be utilized, which allow secure data access, manipulation and evaluation for researchers from different laboratories.