Rapid and effective labeling of brain tissue using TAT-conjugated CdS:Mn/ZnS quantum dots

TAT (a cell penetrating peptide)-conjugated CdSratioMn/ZnS quantum dots (Qdots), intra-arterially delivered to a rat brain, rapidly (within a few minutes) labeled the brain tissue without manipulating the blood-brain-barrier (BBB). Qdot loading was sufficiently high that it allowed a gross fluorescent visualization of the whole rat brain using a low power hand-held UV lamp. Histological data clearly showed that TAT-conjugated Qdots migrated beyond the endothelial cell line and reached the brain parenchyma. Qdots without TAT did not label the brain tissue confirming the fact that TAT peptide was necessary to overcome the BBB. The present study clearly demonstrated the possibility of delivering a large amount of Qdot-based imaging agents to the brain tissue.     

In situ reversible tuning of photoluminescence of Mn2+-doped ZnS quantum dots by redox chemistry

Herein we report the development of a new method for in situ reversible tuning of photoluminescence properties of quantum dots (Qdots) by partial oxidation of population of the emitting species and subsequent chemical reduction of the oxidized form. The concept has been demonstrated using Mn(2+)-doped ZnS Qdots stabilized by acetyl acetonate. Treatment of an aqueous solution of the Qdots (with Mn(OAc)(2) being the source of Mn used for the synthesis of the Qdots) by potassium peroxodisulfate (KPS) led to reduction of intensity of emission due to Mn(2+) ((4)T(1)-(6)A(1)). Subsequent treatment of the solution containing KPS-treated Qdots with NaBH(4) led to regaining of intensity, thus providing reversibility to the tuning, which was possible for more than one cycle. Electron spin resonance (ESR) spectroscopic investigations revealed reduction of the population of Mn(2+) upon treatment with KPS, whereas it went back up upon further treatment with NaBH(4). Interestingly, a mixed population of oxidation states of Mn was indicated to be present in the Qdots prepared using KMnO(4) as the source of Mn. The fluorescence intensity of the Qdots so prepared increased upon treatment with NaBH(4) following synthesis, which was not possible when the source of Mn was Mn(OAc)(2). Transmission electron microscopic and X-ray diffraction studies indicated that oxidation and reduction did not change the sizes of Qdots significantly.     

Surface ion engineering of Mn2+-doped ZnS quantum dots using ion-exchange resins

We report the engineering of surface ions present as defects in doped quantum dots (Qdots) following their synthesis. This was achieved by treating the Qdots with cation-exchange resin beads (CB). An aqueous dispersion of Mn(2+)-doped ZnS Qdots, when treated with different amounts of CB, resulted in two kinds of changes in the emission due to Mn(2+) ions. First, the intensity increased in the presence of a smaller amount of CB, to the extent of a doubled quantum yield. With increased CB as well as incubation time, the emission intensity decreased systematically, accompanied by an increasing blue shift of the peak emission wavelength. Electron spin resonance results indicated the removal of clusters of Mn(2+) present in the Qdots by the CB, which has been attributed to changes in the emission characteristics. Transmission electron microscopy studies revealed that for smaller amounts of CB there was no change in the particle size, whereas for greater amounts the particle size decreased. The results have been explained on the basis of the removal of Mn(2+) (and also Zn(2+)) ions present on the surfaces of Qdots in the form of clusters as well as individual ions.     

Quantum dot-conjugated anti-GRP78 scFv inhibits cancer growth in mice

Semiconductor quantum dots (Qdots) have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78) retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.     

Aptamer-quantum dots conjugates-based ultrasensitive competitive electrochemical cytosensor for the detection of tumor cell

A novel competitive electrochemical cytosensor was reported by using aptamer (Apt)-quantum dots (Qdots) conjugates as a platform for tumor cell recognition and detection. The complementary DNA (cDNA), aptamer and Qdots could be assembled to the gold electrode surface. When the target cells existed, they could compete with cDNA to bind with Apt-Qdots conjugates based on the specific recognition of aptamer to MUC1 protein overexpressed on the cell surface, which resulted in the denaturation of double-stranded DNA structure and the release of the Apt-Qdots conjugates from the electrode. Electrochemical stripping measurement was then employed to determine the Cd²⁺ concentration in Qdots left at the electrode. The peak current was inversely proportional to the logarithmic value of cell concentration ranging from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹ with a detection limit of 100 cells mL⁻¹. Meanwhile, the recognition of aptamer to the target cells could be clearly observed through the strong fluorescence from Qdots. This is an example of the combination of aptamer and nanoparticles for the application of cell analysis, which is essential to cancer diagnosis and therapy.     

A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot–protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins.     

Photocatalytic reduction of an azide-terminated self-assembled monolayer using CdS quantum dots

Ordered, tightly packed aryl-azide-terminated, self-assembled monolayers (SAMs) were created on gold substrates from a new disulfide precursor. These monolayers were reduced at least partially in an aqueous environment using approximately 2 nm CdS quantum dots (Qdots) as photocatalysts to give mixed monolayers of arylamine- and aryl azide-terminated species. The CdS photocatalysts were made available for the reaction by exposure of the azide-terminated SAM to Qdots initially in solution or by preadsorption of the CdS nanoparticles on the SAM. In either case, X-ray photoelectron spectroscopy (XPS), grazing angle Fourier transform infrared spectroscopy (FTIR), and contact angle measurements were used to show the occurrence of the photocatalytic reduction. As further evidence for the presence of arylamine-terminated thiolate in the reduced SAM, these arylamine groups were successfully tagged with fluorescein isothiocyanate (FITC). The use of Qdot photocatalysts to functionalize surfaces may lead to a means to pattern surfaces at the nanoscale.     

CdTe‐paper‐based Visual Sensor for Detecting Methyl Viologen

This study developed a method on detecting methyl viologen (paraquat) using a CdTe‐paper‐based visual sensor. The CdTe Qdots were immobilized on the paper using glycerin. The volume percentages of CdTe in glycerin were optimized to be 50%. The sensing principle is that the methyl viologen quenches the fluorescence intensity of CdTe Qdots in a concentration dependent manner. The sensor is linearly response to the logarithm concentration of the methyl viologen in the range from 0.39 µmol/L to 3.89 mmol/L with a detection limit of 0.16 µmol/L and the correlation coefficient R² of 0.99. Three parallel experiments at the methyl viologen concentration of 38.89 µmol/L give a relative error of 2.45%, which indicates a good reproducibility. The sensor is not disturbed by other pestisides including omethoate, anilofos, machete and glyphosate isopropylamine salt. The advantages of this sensor are disposable, stable, convenient, and easy to operate.     

Quantum dots-based reverse phase protein microarray

CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection. In this study, we established a novel Qdot-based technology platform that can perform accurate and reproducible quantification of protein concentration in a crude cell lysate background. Protein lysates have been spiked with a target protein, and a dilution series of the cell lysate with a dynamic range of three orders of magnitude has been used for this proof-of-concept study. The dilution series has been spotted in microarray format, and protein detection has been achieved with a sensitivity that is at least comparable to standard commercial assays, which are based on horseradish peroxidase (HRP)-catalyzed diaminobenzidine (DAB) chromogenesis. The data obtained through the Qdot method has shown a close linear correlation between relative fluorescence unit and relative protein concentration. The Qdot results are in almost complete agreement with data we obtained with the well-established HRP-DAB colorimetric array (R² = 0.986). This suggests that Qdots can be used for protein quantification in microarray format, using the platform presented here.     

Haloperidol imprinted polymer: preparation,evaluation, and application for drug assay in brain tissue

Several molecularly imprinted polymers (MIPs) were prepared in the present work, and their binding properties were evaluated in comparison with a nonimprinted polymer (NIP). An optimized MIP was selected and applied for selective extraction and analysis of haloperidol in rabbit brain tissue. A molecularly imprinted solid-phase extraction (MISPE) method was developed for cleanup and preconcentration of haloperidol in brain samples before HPLC-UV analysis. Selectivity of the MISPE procedure was investigated using haloperidol and some structurally different drugs with similar polarity that could exist simultaneously in brain tissue. The extraction and analytical process was calibrated in the range of 0.05–10 ppm. The recovery of haloperidol in this MISPE process was calculated between 79.9 and 90.4 %. The limit of detection (LOD) and the limit of quantification (LOQ) of the assay were 0.008 and 0.05 ppm, respectively. Intraday precision and interday precision values for haloperidol analysis were less than 5.86 and 7.63 %, respectively. The MISPE method could effectively extract and concentrate haloperidol from brain tissue in the presence of clozapine and imipramine. Finally, the imprinted polymer was successfully applied for the determination of haloperidol in a real rabbit brain sample after administration of a toxic dose. Therefore, the proposed MISPE method could be applied in the extraction and preconcentration before HPLC-UV analysis of haloperidol in rabbit brain tissue.     

A prodrug of NMDA/glycine site antagonist, L-703,717, with improved BBB permeability: 4-acetoxy derivative and its positron-emitter labeled analog

4-Acetoxy derivative (1) of L-703,717, a high-affinity (IC50=4.5 nM) antagonist for the glycine site of NMDA receptors, was synthesized and its brain uptake was examined using a carbon-11 labeled analog ([11C]1). Initial radioactivity in the brain after intravenous injection of [11C]1 was a 2-fold that of [11C]L-703,717 in mice. Rapid bioconversion of [11C]1 into [11C]L-703,717 was demonstrated by metabolite analyses of rat brain after [11C]1 injection. Ex vivo autoradiography of [11C]1 in rat brain showed the same cerebellar localization of radioactivity as [11C]L-703,717. These results indicate that 1 is a promising pharmacological tool as a prodrug of L-703,717 with improved BBB permeability.     

Azide‐functionalized nanoparticles as quantized building block for the design of soft–soft fluorescent polystyrene core—PAMAM shell nanostructures

This work deals with the covalent coupling of azide‐functionalized polymeric nanoparticles as a reactive core and amino‐terminated PAMAM dendrons as a reactive shell. The nanoassemblies thereby obtained could be modified after the dendronization step by grafting an alkynyl Bodipy dye on the unreacted azide moieties. Only a few steps are required to attain nanoassemblies that could mimic dendrimers of high generation with sizes of nano‐objects beyond those of dendrimers. The structure of the nanoassemblies are composed of a polystyrene core, an inner shell including the Bodipy dyes along with the internal branches of the PAMAM, and the terminal amino groups from the outer shell. The dendritic shell acts as a protective layer that prevents NP from aggregation in a surfactant free aqueous solution. The nano‐objects display absorption and emission maxima above 500 nm with brightness that are the same order of magnitude than Qdots. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 115–126     

Brain regional distribution of the minor and trace elements, Na, K, Sc, Cr, Mn, Co, Zn and Se, in mice bred under Zn-deficient, -adequate and -excessive diets

Instrumental neutron activation analysis was to study the regional distributions of the minor (Na, K) and trace (Sc, Cr, Mn, Co, Zn, Se) elements in mice bred under the controlled diets with Zn content from 0.7 to 3520 ppm in small amounts of brain regions (cerebral cortex, corpus striatum, hippocampus, thalamus and hypothalamus, midbrain, cerebellum, pons and medulla, olfactory bulb). The trace elements were distributed heterogeneously among all brain regions and concentrated in corpus striatum and hippocampus. No significant changes were observed in Zn concentration of most brain regions among the mice bred under Zn-deficient ([Zn] = 3.6 ppm), -adequate and -excessive diets. The results indicate that the homeostasis of Zn seems to be maintained and the brain regional concentrations of Zn seem to be generally constant in all brain regions over a wide range of dietary Zn content from a deficient content as few ppm up to apparently excessive content as 3520 ppm. In an extremely severe Zn-deficient diet ([Zn]<0.7 ppm) where mice can hardly survive, the brain regions may have higher Zn concentration.     

Simultaneous determination of nicotine and its metabolite, cotinine, in rat blood and brain tissue using microdialysis coupled with liquid chromatography: pharmacokinetic application

To elucidate the disposition of nicotine in the brain is important because the neuropharmacological effects from nicotine exposure are centrally predominated. The aim of the present study was to develop a rapid and simple method for the simultaneous determination of unbound nicotine and its main metabolite, cotinine, in rat blood and brain tissue. We coupled a multiple sites microdialysis sampling technique with HPLC-UV system to characterize the pharmacokinetics of both nicotine and cotinine. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and nicotine (2 mg/kg, i.v.) was administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a HPLC system. Both nicotine and cotinine were separated by a phenyl-hexyl column (150 mm x 4.6 mm) from dialysates within 12 min. The mobile phase consisted of an acetonitrile-methanol-20 mM monosodium phosphate buffer (55:45:900, v/v/v, pH adjusted to 5.1) with a flow-rate of 1 ml/min. The wavelength of the UV detector was set at 260 nm. The limit of quantification for nicotine and cotinine were 0.25 microg/ml and 0.05 microg/ml, respectively. Intra- and inter-day precision and accuracy of both measurements fell well within the predefined limits of acceptability. The blood and brain concentration-time profile of nicotine and cotinine suggests that nicotine is easily to get into the central nervous system and cotinine exhibits a long retention time and accumulates in blood.     

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot–MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen–antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC₅₀) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10–100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1–9.2%. Figure Shematic diagram of the direct cFLISA procedure Jianzhong Shen and Fei Xu contributed equally to this work.     

NORMAL BRAIN TISSUE RESPONSE TO PHOTODYNAMIC THERAPY: HISTOLOGY, VASCULAR PERMEABILITY AND SPECIFIC GRAVITY

The response of photodynamic therapy on normal brain was investigated in 140 Fisher rats. The rats were injected i.p. with Photofrin II (12.5 mg/kg) and 48 h later the dural area over the frontal cortex was photoactivated with red light (630 +/- 1 nm) from an argon dye laser. Treatment was performed with optical energy densities of 140 and 70 J/cm2. Histopathology, vascular permeability and specific gravity measurements were conducted on different populations of rats at 4 h, 24 h, 72 h and 1 week after photodynamic therapy (PDT). Histopathology revealed similar gross and microscopic pathology associated with light energies of 70 and 140 J/cm2 after all time points. A large cerebral infarct approximately the size of the brain surface area treated, evolved 24 h following treatment. Evans blue extravasation indicated a small area of vascular permeability evident as early as 4 h following PDT treatment at both energy levels, with increasing permeability evident at later time points. Specific gravity measurements taken on a representative area of the lesion indicated a significant (P less than 0.01) amount of edema present at 24 h post treatment with a gradual reduction approaching control values over the time period of 1 week. The data indicate a significant amount of damage to normal brain from low PDT treatment doses.     

PHOTODYNAMIC THERAPY OF MALIGNANT PRIMARY BRAIN TUMOURS: CLINICAL EFFECTS, POSTOPERATIVE ICP, and LIGHT PENETRATION OF THE BRAIN

Abstract We are reporting our experience with intraoperative PDT in 32 patients with malignant supratentorial gliomas; in 19 cases the tumour was recurrent. There were 20 males and 12 females with an age range of 17-73 (mean = 45) yr. The first 8 patients in this series received HpD (Photofrin I) and the next 24 received DHE (Photofrin II). A photo-illuminating device, of the author's design, was coupled to an argon dye pump laser in order to deliver light at 630 nm to a tumour cavity created by radical tumour resection and/or tumour cyst drainage. The total light energy delivered ranged from 440 to 3888 J and the light energy density ranged from 8 to 68 J cm^?2. There were two post-operative deaths as the consequence of hematoma accumulation in an extensive tumour resection cavity. In two patients neurological function was worse post-operatively and did not recover. Post-operative cerebral edema was pronounced in eight cases and required emergency craniotomy in two patients (the histology from both showed hemorrhagic necrosis of residual tumour). We have fashioned continuous post-operative ICP measurements in the last 15 patients treated with PDT and compared the values to those obtained from cases with malignant gliomas who did not have PDT; the mean ICP was significantly greater in the PDT group. Four patients developed wound infections; two of these required surgical treatment. Four patients, two of whom were hemiparetic, developed deep vein thrombosis and required anticoagulant therapy. There were no adverse systemic reactions to the administration of either photosensitizer and only 3 skin photosensitivity reactions. Follow up has ranged from 1 to 26 months at the time of writing; 38% were still alive; in the interval between PDT and death, the deaths per observation year was 1.11 for the whole group and 1.00 when the two post-operative deaths are excluded. In the interval between first diagnosis and death the rate was 0.45 deaths per observation year. Photodynamic therapy of malignant brain tumours using surface or cavitary photo-illumination can be carried out with acceptable risk. In eight patients we determined the penetration depth of light in brain tissue in vivo by reading the detected light flux from a fiber passed radially into the brain towards the centre of the irradiation volume. The optical fiber consisted of a single 400 μ.m, cleaved fiber fixed in a 17-gauge biopsy needle coupled to a photometer. The light penetration depth ranged from 0.8 to 4.9. The mean PD values in the‘tumour’group and the‘brain’group was 2.9 ± 1.5 and 1.5 ± 0.43, respectively. We attribute this significant difference to the differences in the absorption and scattering properties of brain and tumour.     

Studies on Neurosteroids XXI: an improved liquid chromatography-tandem mass spectrometric method for determination of 5alpha-androstane-3alpha,17beta-diol in rat brains.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of the rat brain 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol) has been developed and validated. The brain extract was purified using solid-phase extraction cartridges, derivatized with isonicotinoyl azide, and subjected to LC-MS/MS. The method was accurate and reproducible, and the limit of quantitation was 0.1 ng/g tissue when a 100-mg tissue sample was used. The change in the brain 3alpha,5alpha-Adiol level by immobilization stress was also analyzed using the developed method.     

Determination of AJ-3941, a possible agent for the treatment of cerebrovascular disorders, in plasma and brain by means of high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method with fluorescence detection is described for the determination of AJ-3941 (I), a possible agent for the treatment of cerebrovascular disorders, in plasma and brain tissue. A simple hexane extraction was used for plasma, and for brain homogenate the hexane extract was further purified by solid-phase extraction. The determination limit was ca. 3 ng/ml for both plasma (0.5 ml) and 10% (w/v) brain homogenate (1 ml). The method was applied to the determination of I in plasma and brain samples of experimental animals.