Carbon nanotube bottles for incorporation, release and enhanced cytotoxic effect of cisplatin.

Carbon nanotubes (CNTs) have emerged as promising drug delivery systems particularly for cancer therapy, due to their abilities to overcome some of the challenges faced by cancer treatment, namely non-specificity, poor permeability into tumour tissues, and poor stability of anticancer drugs. Encapsulation of anticancer agents inside CNTs provides protection from external deactivating agents. However, the open ends of the CNTs leave the encapsulated drugs exposed to the environment and eventually their uncontrolled release before reaching the desired target. In this study, we report the successful encapsulation of cisplatin, a FDA-approved chemotherapeutic drug, into multi-walled carbon nanotubes and the capping at the ends with functionalised gold nanoparticles to achieve a "carbon nanotube bottle" structure. In this proof-of-concept study, these caps did not prevent the encapsulation of drug in the inner space of CNTs; on the contrary, we achieved higher drug loading inside the nanotubes in comparison with data reported in literature. In addition, we demonstrated that encapsulated cisplatin could be delivered in living cells under physiological conditions to exert its pharmacological action.

Spatial memory training induces morphological changes detected by manganese-enhanced MRI in the hippocampal CA3 mossy fiber terminal zone.

Hippocampal mossy fibers (MFs) can show plasticity of their axon terminal arbor consequent to learning a spatial memory task. Such plasticity is seen as translaminar sprouting from the stratum lucidum (SL) of CA3 into the stratum pyramidale (SP) and the stratum oriens (SO). However, the functional role of this presynaptic remodeling is still obscure. In vivo imaging that allows longitudinal observation of such remodeling could provide a deeper understanding of this presynaptic growth phenomenon as it occurs over time. Here we used manganese-enhanced magnetic resonance imaging (MEMRI), which shows a high-contrast area that co-localizes with the MFs. This technique was applied in the detection of learning-induced MF plasticity in two strains of rats. Quantitative analysis of a series of sections in the rostral dorsal hippocampus showed increases in the CA3a' area in MEMRI of trained Wistar rats consistent with the increased SO+SP area seen in the Timm's staining. MF plasticity was not seen in the trained Lister-Hooded rats in either MEMRI or in Timm's staining. This indicates the potential of MEMRI for revealing neuro-architectures and plasticity of the hippocampal MF system in vivo in longitudinal studies.

Structural basis for the interaction of unstructured neuron specific substrates neuromodulin and neurogranin with Calmodulin.

Neuromodulin (Nm) and neurogranin (Ng) are neuron-specific substrates of protein kinase C (PKC). Their interactions with Calmodulin (CaM) are crucial for learning and memory formation in neurons. Here, we report the structure of IQ peptides (24aa) of Nm/Ng complexed with CaM and their functional studies with full-length proteins. Nm/Ng and their respective IQ peptides are intrinsically unstructured; however, upon binding with CaM, IQ motifs adopt a helical conformation. Ser41 (Ser36) of Nm (Ng) is located in a negatively charged pocket in the apo CaM and, when phosphorylated, it will repel Nm/Ng from CaM. These observations explain the mechanism by which PKC-induced Ser phosphorylation blocks the association of Nm/Ng with CaM and interrupts several learning- and memory-associated functions. Moreover, the present study identified Arg as a key CaM interacting residue from Nm/Ng. This residue is crucial for CaM-mediated function, as evidenced by the inability of the Ng mutant (Arg-to-Ala) to potentiate synaptic transmission in CA1 hippocampal neurons.

Graphene versus Multi-Walled Carbon Nanotubes for Electrochemical Glucose Biosensing.

: A simple procedure was developed for the fabrication of electrochemical glucose biosensors using glucose oxidase (GOx), with graphene or multi-walled carbon nanotubes (MWCNTs). Graphene and MWCNTs were dispersed in 0.25% 3-aminopropyltriethoxysilane (APTES) and drop cast on 1% KOH-pre-treated glassy carbon electrodes (GCEs). The EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide)-activated GOx was then bound covalently on the graphene- or MWCNT-modified GCE. Both the graphene- and MWCNT-based biosensors detected the entire pathophysiological range of blood glucose in humans, 1.4-27.9 mM. However, the direct electron transfer (DET) between GOx and the modified GCE's surface was only observed for the MWCNT-based biosensor. The MWCNT-based glucose biosensor also provided over a four-fold higher current signal than its graphene counterpart. Several interfering substances, including drug metabolites, provoked negligible interference at pathological levels for both the MWCNT- and graphene-based biosensors. However, the former was more prone to interfering substances and drug metabolites at extremely pathological concentrations than its graphene counterpart.

Mediatorless amperometric glucose biosensing using 3-aminopropyltriethoxysilane-functionalized graphene.

A mediatorless glucose biosensor was developed by the immobilization of glucose oxidase (GOx) to graphene-functionalized glassy carbon electrode (GCE). The surface of GCE was functionalized with graphene by incubating it with graphene dispersed in 3-aminopropyltriethoxysilane (APTES), which acted both as a dispersion agent for graphene and as an amine surface modification agent for GCE and graphene. This was followed by the covalent binding of GOx to graphene-functionalized GCE using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) based crosslinking. Graphene provided signal enhancement by providing greater surface area for GOx binding, while APTES-functionalization led to a higher GOx immobilization density by providing free amino groups for crosslinking. The developed biosensor used a redox potential of -0.45 V (vs. Ag/AgCl) for detecting glucose in the diabetic pathophysiological range 0.5-32 mM. There was no interference from endogenous electroactive substances and drug metabolites. The developed biosensor was further validated for detecting blood glucose in commercial artificial blood glucose linearity standards in the range 1.4-27.9 mM. Therefore, it is ideal for diabetic blood glucose monitoring. The developed bioanalytical procedure for preparation of GOx-bound graphene-functionalized GCEs had high production reproducibility and high storage stability, which is appropriate for the commercial mass production of enzyme-bound electrodes.

Rapid and simple preparation of a reagentless glucose electrochemical biosensor.

A rapid and simple procedure was developed for the preparation of a highly stable and leach-proof glucose oxidase (GOx)-bound glassy carbon electrode (GCE). Crosslinked GOx via glutaraldehyde was drop-cast on a KOH-pretreated GCE followed by drop-casting of 3-aminopropyltriethoxysilane (APTES) to form a stable bioactive layer. At -0.45 V, the biosensor exhibited a wide dynamic detection range of 0.5-48 mM for commercial glucose and 1.3-28.2 mM for Sugar-Chex blood glucose linearity standards. Several endogenous electroactive substances and drug metabolites commonly found in blood were tested and provoked no signal response. To our knowledge, the developed procedure is the most rapid method for preparing a glucose biosensor. The biosensor suffered no biofouling after 7 days of immersion in Sugar-Chex blood glucose. With excellent production reproducibility, GOx-bound electrodes stored dry at room temperature retained their initial activity after several weeks.

Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy.

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (µg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240±9µM in wet weight level (135±5µg/g dry weight) in the stratum lucidum (SL) compared to 144±6µM in wet weight level (81±3µg/g dry weight) in the stratum pyramidale (SP) and 78±10µM in wet weight level (44±5µg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

Technology behind commercial devices for blood glucose monitoring in diabetes management: a review.

The blood glucose monitoring devices (BGMDs) are an integral part of diabetes management now-a-days. They have evolved tremendously within the last four decades in terms of miniaturization, rapid response, greater specificity, simplicity, minute sample requirement, painless sample uptake, sophisticated software and data management. This article aims to review the developments in the technologies behind commercial BGMD, especially those in the areas of chemistries, mediators and other components. The technology concerns, on-going developments and future trends in blood glucose monitoring (BGM) are also discussed.

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay.

This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu(2+) in the BCA Kit's reagent B (4% cupric sulfate) in a manner similar to that of the protein.

Purification, functionalization, and bioconjugation of carbon nanotubes.

Bioconjugation of carbon nanotubes (CNTs) with biomolecules promises exciting applications such as biosensing, nanobiocomposite formulation, design of drug vector systems, and probing protein interactions. Pristine CNTs, however, are virtually water-insoluble and difficult to evenly disperse in a liquid matrix. Therefore, it is necessary to attach molecules or functional groups to their sidewalls to enable bioconjugation. Both noncovalent and covalent procedures can be used to conjugate CNTs with a target biomolecule for a specific bioapplication. This chapter presents a few selected protocols that can be performed at any wet chemistry laboratory to purify and biofunctionalize CNTs. The preparation of CNTs modified with metallic nanoparticles, especially gold, is also described since biomolecules can bind and self-organize on the surfaces of such metal-decorated CNTs.

Advances in carbon nanotube based electrochemical sensors for bioanalytical applications.

Electrochemical (EC) sensing approaches have exploited the use of carbon nanotubes (CNTs) as electrode materials owing to their unique structures and properties to provide strong electrocatalytic activity with minimal surface fouling. Nanofabrication and device integration technologies have emerged along with significant advances in the synthesis, purification, conjugation and biofunctionalization of CNTs. Such combined efforts have contributed towards the rapid development of CNT-based sensors for a plethora of important analytes with improved detection sensitivity and selectivity. The use of CNTs opens an opportunity for the direct electron transfer between the enzyme and the active electrode area. Of particular interest are also excellent electrocatalytic activities of CNTs on the redox reaction of hydrogen peroxide and nicotinamide adenine dinucleotide, two major by-products of enzymatic reactions. This excellent electrocatalysis holds a promising future for the simple design and implementation of on-site biosensors for oxidases and dehydrogenases with enhanced selectivity. To date, the use of an anti-interference layer or an artificial electron mediator is critically needed to circumvent unwanted endogenous electroactive species. Such interfering species are effectively suppressed by using CNT based electrodes since the oxidation of NADH, thiols, hydrogen peroxide, etc. by CNTs can be performed at low potentials. Nevertheless, the major future challenges for the development of CNT-EC sensors include miniaturization, optimization and simplification of the procedure for fabricating CNT based electrodes with minimal non-specific binding, high sensitivity and rapid response followed by their extensive validation using "real world" samples. A high resistance to electrode fouling and selectivity are the two key pending issues for the application of CNT-based biosensors in clinical chemistry, food quality and control, waste water treatment and bioprocessing.

Interfacing carbon nanotubes with living mammalian cells and cytotoxicity issues.

The unique structures and properties of carbon nanotubes (CNTs) have attracted extensive investigations for many applications, such as those in the field of biomedical materials and devices, biosensors, drug delivery, and tissue engineering. Anticipated large-scale productions for numerous diversified applications of CNTs might adversely affect the environment and human health. For successful applications in the biomedical field, the issue of interfacing between CNTs and mammalian cells in vitro needs to be addressed before in vivo studies can be carried out systematically. We review the important studies pertaining to the internalization of CNTs into the cells and the culturing of cells on the CNT-based scaffold or support materials. The review will focus on the description of a variety of factors affecting CNT cytotoxicity: type of CNTs, impurities, lengths of CNTs, aspect ratios, dispersion, chemical modification, and assaying methods of cytotoxicity.

Modification of carbon nanotubes with redox hydrogel: improvement of amperometric sensing sensitivity for redox enzymes.

This study demonstrated that redox hydrogel-modified carbon nanotube (CNT) electrodes can be developed as an amperometric sensor that are sensitive, specific and fast and do not require auxiliary enzymes. A redox polymer, poly(vinylimidazole) complexed with Os(4,4'-dimethylbpy)(2)Cl (PVI-dmeOs) was electrodeposited on Ta-supported multi-walled CNTs. The resulted PVI-dmeOs thin film did not change the surface morphology of the CNTs, but turned the CNT surface from hydrophobic to hydrophilic, as studied by scanning electron microscopy (SEM) and static water contact angle measurements. Cyclic voltammetry measurements in a Fe(CN)(6)(3-) solution and electrochemical impedance measurements in an equimolar Fe(CN)(6)(3-/4-) solution demonstrated that the PVI-dmeOs hydrogel thin film was electronic conductive with a resistance of about 15Omega. The PVI-dmeOs/CNT electrodes sensed rapidly, sensitively and specifically to model redox enzymes (glucose oxidase (GOD) and lactate oxidase (LOD)) in amperometric experiments in electrolyte solutions containing the substrates of the measured redox enzymes. Both the CNT substrate and the thin PVI-dmeOs film enhanced the sensing sensitivities. Exploration of the mechanisms suggests that the PVI-dmeOs film may enhance the sensing sensitivities by wiring the enzyme molecules through the redox centers tethered on the mobile redox polymer backbones to the CNT electrodes.

Characterization and field emission performance of electrochemically synthesized FeOOH nanowalls.

We report a direct method for the synthesis of Iron(III) oxyhydroxide (FeOOH) nanowalls using an electrochemical technique at room temperature. The length of the nanowalls can be varied depending on the number of repetitive potentiostatic pulse cycles during the electrochemical process. The samples were characterized by ex-situ techniques such as SEM, XPS, FTIR, and TEM. Field emission performances of these nanowalls are also reported for the first time. The measured turn-on electric field is about 4.8 V/microm, with emission current density of 0.12 mA/cm2 at 7.3 V/microm. This technique provides a simple alternative method for large area synthesis of FeOOH nanowalls.

Selective and sensitive electrochemical detection of glucose in neutral solution using platinum-lead alloy nanoparticle/carbon nanotube nanocomposites.

Electrodeposition of Pt-Pb nanoparticles (PtPbNPs) to multi-walled carbon nanotubes (MWCNTs) resulted in a stable PtPbNP/MWCNT nanocomposite with high electrocatalytic activity to glucose oxidation in either neutral or alkaline medium. More importantly, the nanocomposite electrode with a slight modification exhibited high sensitivity, high selectivity, and low detection limit in amperometric glucose sensing at physiological neutral pH (poised at a negative potential). At +0.30 V in neutral solution, the nanocomposite electrode exhibited linearity up to 11 mM of glucose with a sensitivity of 17.8 microA cm(-2) mM(-1) and a detection limit of 1.8 microM (S/N=3). Electroactive ascorbic acid (0.1 mM), uric acid (0.1 mM) and fructose (0.3 mM) invoked only 23%, 14% and 9%, respectively, of the current response obtained for 3 mM glucose. At -0.15 V in neutral solution, the electrode responded linearly to glucose up to 5 mM with a detection limit of 0.16 mM (S/N=3) and detection sensitivity of approximately 18 microA cm(-2) mM(-1). At this negative potential, ascorbic acid, uric acid, and fructose were not electroactive, therefore, not interfering with glucose sensing. Modification of the nanocomposite electrode with Nafion coating followed by electrodeposition of a second layer of PtPbNPs on the Nafion coated PtPbNP/MWCNT nanocomposite produced a glucose sensor (poised at -0.15 V) with a lower detection limit (7.0 microM at S/N=3) and comparable sensitivity, selectivity and linearity compared to the PtPbNP/MWCNT nanocomposite. The Nafion coating lowered the detection limit by reducing the background noise, while the second layer of PtPbNPs restored the sensitivity to the level before Nafion coating.

Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation.

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.

Frontal cortical alpha7 and alpha4beta2 nicotinic acetylcholine receptors in working and reference memory.

The alpha7 and alpha4beta2 nicotinic acetylcholine receptor (nAchR) subtypes have been shown to be involved in memory. It is also known that losses of frontal cortical nAchRs are correlated to declining memory function in Alzheimer's disease, but the subtype-specific role of frontal cortical nAchRs in memory has not been well characterized. Hence, we sought to understand the role of frontal cortical alpha7 and alpha4beta2 nAchR subtypes in both working and reference memory by observing the effects of subtype specific agonists and antagonists on radial arm maze performance. It was found that alpha7 nAchRs in the frontal cortex are involved in working and reference memory, while alpha4beta2 nAchRs are only involved in working memory. Throughout the study, drug treatments did not affect motor functionality in the animals. Our data thus sheds further light on the frontal cortex as an important anatomical locus for nAchR-mediated memory function in the brain, and highlights the differing role of alpha7 and alpha4beta2 nAchRs in long and short term memory.

Tanshinone IIB, a primary active constituent from Salvia miltiorrhza, exhibits neuro-protective activity in experimentally stroked rats.

Tanshinone IIB (TSB) is a major active constituent of the root of Salvia miltiorrhiza (Danshen) used in the treatment of acute stroke. Danshen extracts and TSB have shown marked neuron-protective effects in mouse studies but there is a lack of clinical evidence for the neuron-protective effects of Danshen and its active ingredients. This study investigated the neuron-protective effects of TSB in experimentally stroked rats. TSB at 5 and 25 mg/kg by intraperitoneal injection significantly reduced the focal infarct volume, cerebral histological damage and apoptosis in rats subjected to middle cerebral artery occlusion (MCAO) compared to MCAO rats receiving vehicle. This study demonstrated that TSB was effective in reducing stroke-induced brain damage and may represent a novel drug candidate for further development. Further mechanistic studies are needed for the neuron-protective activity of TSB.

Cell adhesion properties on photochemically functionalized diamond.

The biocompatibility of diamond was investigated with a view toward correlating surface chemistry and topography with cellular adhesion and growth. The adhesion properties of normal human dermal fibroblast (NHDF) cells on microcrystalline and ultrananocrystalline diamond (UNCD) surfaces were measured using atomic force microscopy. Cell adhesion forces increased by several times on the hydrogenated diamond surfaces after UV irradiation of the surfaces in air or after functionalization with undecylenic acid. A direct correlation between initial cell adhesion forces and the subsequent cell growth was observed. Cell adhesion forces were observed to be strongest on UV-treated UNCD, and cell growth experiments showed that UNCD was intrinsically more biocompatible than microcrystalline diamond surfaces. The surface carboxylic acid groups on the functionalized diamond surface provide tethering sites for laminin to support the growth of neuron cells. Finally, using capillary injection, a surface gradient of polyethylene glycol could be assembled on top of the diamond surface for the construction of a cell gradient.

Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress.

Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca(2+)/CaM complex and modulates the homeostasis of intracellular calcium in neurons. Previous work showed Ng oxidation by NO donor induces increase in [Ca(2+)](i). The current study demonstrated that the gene transcription of Ng could be up-regulated by various nitric oxide (NO) donors via a NO-soluble guanylyl cyclase (sGC)-mediated pathway. Furthermore, ectopic expression of neuronal nitric oxide synthase (nNOS) in human embryonic kidney 293 cells (HEK 293) exhibited a nNOS-concentration-dependent biphasic regulatory effect on Ng gene transcription. One of the NO donors, sodium nitroprusside (SNP), however, induced cell death of neuroblastoma Neuro-2a cells. The potency of SNP-induced cell death was shown to be higher in Neuro-2a cells expressing recombinant Ng, as compared with Neuro-2a control cells without Ng expression in cell viability and apoptosis assays. Single-cell fluorescence imaging and site-directed mutagenesis studies suggest that Ng promotes SNP-induced cell death through an amplification of calcium-mediated signaling, which requires the interaction between CaM and IQ motif of Ng. Increased neuronal susceptibility rendered by Ng in response to pathophysiological NO production is suggested to be involved in the selective vulnerability of neurons to oxidative insults in the CNS.