Improving Tumor Accumulation of Aptamers by Prolonged Blood Circulation.

Developing cancer targeted medicine depends on increasing delivery efficiency and tumor site accumulation of theranostic agents. To accomplish this, we report a modification of PTK7 receptor-specific aptamer Sgc8 with the small molecule Evans Blue (EB), thus implementing an albumin binding hitchhike strategy for prolonged blood circulation. The EB molecule could insert into the hydrophobic region of serum albumin and form an aptamer/albumin complex. This complex showed superior physiological stability, facilitating longer blood half-life, and maintaining its targeting capacity. Successful conjugation of EB-aptamers was confirmed by a series of characterization methods. Targeting performance was tested on a xenografted mouse tumor model. Taking advantage of the long circulating aptamer/HSA complex, improved accumulation, and delivery efficiency to the tumor site were achieved. Through ex vivo quantification of the EB-Sgc8 aptamers' biodistribution, the mechanism of improved targeting performance was illuminated. Therefore, the increased aptamers tumor delivery efficiency and accumulation indicate that prolonging blood circulation is a promising strategy to improve aptamers' targeted delivery performance in the future clinical translation.

MRI of Whole Rat Brain Perivascular Network Reveals Role for Ventricles in Brain Waste Clearance.

Investigating the mechanisms by which metabolic wastes are cleared from nervous tissue is important for understanding natural function and the pathophysiology of several neurological disorders including Alzheimer's disease. Recent evidence suggests clearance may be the function of annular spaces around cerebral blood vessels, called perivascular spaces (PVS), through which cerebrospinal fluid (CSF) is transported from the subarachnoid space into brain parenchyma to exchange with interstitial fluid (also known as the glymphatic system). In this work, an MRI-based methodology was developed to reconstruct the PVS network in whole rat brain to better elucidate both PVS uptake and clearance pathways. MR visible tracer (Gd-albumin) was infused in vivo into the CSF-filled lateral ventricle followed by ex vivo high-resolution MR imaging at 17.6 T with an image voxel volume two orders of magnitude smaller than previously reported. Imaged tracer distribution patterns were reconstructed to obtain a more complete brain PVS network. Several PVS connections were repeatedly highlighted across different animals, and new PVS connections between ventricles and different parts of the brain parenchyma were revealed suggesting a possible role for the ventricles as a source or sink for solutes in the brain. In the future, this methodology may be applied to understand changes in the PVS network with disease.

Amyloids, Congo red and the apple-green effect.

This paper attempts to find evidence of the previously proposed opinion that amyloids complex with Congo red molecules which preserve their supramolecular organization. As evidence of the overpowering tendency of Congo red molecules to self-assemble, we present an increasing acidity of molecules that follows increasing concentration of the dye, and a highly notable nonlinear increase in absorbance in the UV band (300-400 nm). This effect is analyzed in a model where the amyloid fibril is simulated by polyvinyl alcohol, providing a scaffold to stabilize a long Congo red micelle. Enormous absorbance in the UV band, coupled with the increasing association capabilities of individual Congo red molecules may cause the absorbance to extend even into the visible band. In addition, the UV and visual absorbance bands shift significantly, depending on conditions, and may either approach or recede from each other, leading to spectral changes which may be observed under polarized light. This commonly observed spectral variability appears to be associated with the strong capacity for electron delocalization in supramolecular Congo red complexed with amyloids.

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse.

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

Structural insights into pH-responsive drug release of self-assembling human serum albumin-silk fibroin nanocapsules.

Inflammation processes are associated with significant decreases in tissue or lysosomal pH from 7.4 to 4, a fact that argues for the application of pH-responsive drug delivery systems. However, for their design and optimization a full understanding of the release mechanism is crucial. In this study we investigated the pH-depending drug release mechanism and the influence of silk fibroin (SF) concentration and SF degradation degree of human serum albumin (HSA)-SF nanocapsules. Sonochemically produced nanocapsules were investigated regarding particle size, colloidal stability, protein encapsulation, thermal stability and drug loading properties. Particles of the monodisperse phase showed average hydrodynamic radii between 438 and 888 nm as measured by DLS and AFM and a zeta potential of -11.12 ±â€¯3.27 mV. Together with DSC results this indicated the successful production of stable nanocapsules. ATR-FTIR analysis demonstrated that SF had a positive effect on particle formation and stability due to induced beta-sheet formation and enhanced crosslinking. The pH-responsive release was found to depend on the SF concentration. In in-vitro release studies, HSA-SF nanocapsules composed of 50% SF showed an increased pH-responsive release for all tested model substances (Rhodamine B, Crystal Violet and Evans Blue) and methotrexate at the lowered pH of 4.5 to pH 5.4, while HSA capsules without SF did not show any pH-responsive drug release. Mechanistic studies using confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS) analyses showed that increases in particle porosity and decreases in particle densities are directly linked to pH-responsive release properties. Therefore, the pH-responsive release mechanism was identified as diffusion controlled in a novel and unique approach by linking scattering results with in-vitro studies. Finally, cytotoxicity studies using the human monocytic THP-1 cell line indicated non-toxic behavior of the drug loaded nanocapsules when applied in a concentration of 62.5 µg mL-1. Based on the obtained release properties of HSA-SF nanocapsules formulations and the results of in-vitro MTT assays, formulations containing 50% SF showed the highest requirements arguing for future in vivo experiments and application in the treatment of inflammatory diseases.

Niosomes decorated with dual ligands targeting brain endothelial transporters increase cargo penetration across the blood-brain barrier.

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles.

Subtle thinning of retinal layers without overt vascular and inflammatory alterations in a rat model of prediabetes.

Purpose: Diabetic retinopathy is a neurovascular disease characterized by increased permeability of the blood-retinal barrier, changes in the neural components of the retina, and low-grade chronic inflammation. Diabetic retinopathy is a major complication of diabetes; however, the impact of a prediabetic state on the retina remains to be elucidated. The aim of this study was to assess possible early retinal changes in prediabetic rats, by evaluating changes in the integrity of the blood-retinal barrier, the retinal structure, neural markers, and inflammatory mediators. Methods: Several parameters were analyzed in the retinas of Wistar rats that drank high sucrose (HSu; 35% sucrose solution during 9 weeks, the prediabetic animal model) and were compared with those of age-matched controls. The permeability of the blood-retinal barrier was assessed with the Evans blue assay, and the content of the tight junction proteins and neural markers with western blotting. Optical coherence tomography was used to evaluate retinal thickness. Cell loss at the ganglion cell layer was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and by evaluating the immunoreactivity of the Brn3a transcription factor. To assess retinal neuroinflammation, the mRNA expression and protein levels of inducible nitric oxide synthase isoform (iNOS), interleukin-1 beta (IL-1ß), and tumor necrosis factor (TNF) were evaluated. Iba1 and MHC-II immunoreactivity and translocator protein (TSPO) mRNA levels were assessed to study the microglial number and activation state. Results: The thickness of the inner retinal layers of the HSu-treated animals decreased. Nevertheless, no apoptotic cells were observed, and no changes in retinal neural markers were detected in the retinas of the HSu-treated animals. No changes were detected in the permeability of the blood-retinal barrier, as well as the tight junction protein content between the HSu-treated rats and the controls. In addition, the inflammatory parameters remained unchanged in the retina despite the tendency for an increase in the number of retinal microglial cells. Conclusions: In a prediabetic rat model, the retinal structure is affected by the thinning of the inner layers, without overt vascular and inflammatory alterations. The results suggest neuronal dysfunction (thinning of the inner retina) that may precede or anticipate the vascular and inflammatory changes. Subtle structural changes might be viewed as early disturbances in an evolving disease, suggesting that preventive strategies (such as the modification of diet habits) could be applied at this stage, before the progression toward irreversible dysfunction and damage to the retina.

Radioligand Therapy of Prostate Cancer with a Long-Lasting Prostate-Specific Membrane Antigen Targeting Agent 90Y-DOTA-EB-MCG.

Several radioligands targeting prostate-specific membrane antigen (PSMA) have been clinically introduced as a new class of radiotheranostics for the treatment of prostate cancer. Among them, ((( R)-1-carboxy-2-mcercaptoethyl)carbamoyl)-l-glutamic acid (MCG) has been successfully labeled with radioisotopes for prostate cancer imaging. The aim of this study is to conjugate MCG with an albumin binding moiety to further improve the in vivo pharmacokinetics. MCG was conjugated with an Evans blue (EB) derivative for albumin binding and a DOTA chelator. PSMA positive (PC3-PIP) and PSMA negative (PC3) cells were used for both in vitro and in vivo studies. Longitudinal PET imaging was performed at 1, 4, 24, and 48 h post-injection to evaluate the biodistribution and tumor uptake of 86Y-DOTA-EB-MCG. DOTA-EB-MCG was also labeled with 90Y for radionuclide therapy. Besides tumor growth measurement, tumor response to escalating therapeutic doses were also evaluated by immunohistochemistry and fluorescence microscopy. Based on quantification from 86Y-DOTA-EB-MCG PET images, the tracer uptake in PC3-PIP tumors increased from 22.33 ± 2.39%ID/g at 1 h post-injection (p.i.), to the peak of 40.40 ± 4.79%ID/g at 24 h p.i. Administration of 7.4 MBq of 90Y-DOTA-EB-MCG resulted in significant regression of tumor growth in PSMA positive xenografts. No apparent toxicity or body weight loss was observed in all treated mice. Modification of MCG with an Evans blue derivative resulted in a highly efficient prostate cancer targeting agent (EB-MCG), which showed great potential in prostate cancer treatment after being labeled with therapeutic radioisotopes.

Novel Structural Modification Based on Evans Blue Dye to Improve Pharmacokinetics of a Somastostatin-Receptor-Based Theranostic Agent.

The development of somastatin (SS) peptide analogues for the detection and treatment of neuroendocrine tumors has been successful with the recent FDA approval of 68Ga-DOTA-TATE and 177Lu-DOTA-TATE. The structure of these peptide constructs contains the peptide binding motif that binds to the receptor with high affinity, a chelator to complex the radioactive metal, and a linker between the peptide and chelator. However, these constructs suffer from rapid blood clearance, which limits their tumor uptake. In this study, this design has been further improved by incorporating a modification to control the in vivo pharmacokinetics. Adding a truncated Evans Blue (EB) dye molecule into the construct provides a prolonged half-life in blood as a result of its low micromolar affinity to albumin. We compared 177Lu-DOTA-TATE to the modified 177Lu Evans Blue compound (177Lu-DMEB-TATE), in vitro and in vivo in mice bearing A427-7 xenografts. The tumor uptake of 177Lu-DMEB-TATE was significantly greater than the uptake of 177Lu-DOTA-TATE in the biodistribution and SPECT-imaging studies. The therapeutic effect of the 177Lu-DMEB-TATE construct was superior to the that of the 177Lu-DOTA-TATE construct at the doses evaluated.

Photoacoustic imaging of lymphatic pumping.

The lymphatic system is responsible for fluid homeostasis and immune cell trafficking and has been implicated in several diseases, including obesity, diabetes, and cancer metastasis. Despite its importance, the lack of suitable in vivo imaging techniques has hampered our understanding of the lymphatic system. This is, in part, due to the limited contrast of lymphatic fluids and structures. Photoacoustic imaging, in combination with optically absorbing dyes or nanoparticles, has great potential for noninvasively visualizing the lymphatic vessels deep in tissues. Multispectral photoacoustic imaging is capable of separating the components; however, the slow wavelength switching speed of most laser systems is inadequate for imaging lymphatic pumping without motion artifacts being introduced into the processed images. We investigate two approaches for visualizing lymphatic processes in vivo. First, single-wavelength differential photoacoustic imaging is used to visualize lymphatic pumping in the hindlimb of a mouse in real time. Second, a fast-switching multiwavelength photoacoustic imaging system was used to assess the propulsion profile of dyes through the lymphatics in real time. These approaches may have profound impacts in noninvasively characterizing and investigating the lymphatic system.

Enhanced podocyte vesicle transport in the nephrotic rat.

Albumin endocytosis is enhanced in the podocytes of minimal change nephrotic syndrome. We investigated that the endocytic vesicle transport in the podocyte using three-dimensional observation in a rat model of puromycin aminonucleoside (PAN)-induced nephrotic syndrome. At day 7, Evans Blue-labeled albumin was intravenously injected in PAN rats, and one kidney was fixed for a morphological analysis; the other was used for the isolation of glomeruli through sieving and protein analyses. Evans Blue-labeled albumin was found to accumulate in an increased number of vesicles in the podocytes of PAN rat. Continuous sections and its three-dimensional observation demonstrated that vesicles may be transported from the cytoplasm to the apical membrane of the podocytes. The increased protein bands in the gel electrophoresis of the sieved glomeruli of nephrotic rats were analyzed by mass spectrometry in comparison to the control rats. The major proteins increased in the nephrotic rats were cytoplasmic dynein 1 heavy chain, myosin IX, and myosin VIIb. In conclusion, the podocyte endocytic vesicles carrying albumin increased with glomerular cytoplasmic dynein and myosin in minimal change nephrotic rats.

Novel "Add-On" Molecule Based on Evans Blue Confers Superior Pharmacokinetics and Transforms Drugs to Theranostic Agents.

One of the major design considerations for a drug is its pharmacokinetics in the blood. A drug with a short half-life in the blood is less available at a target organ. Such a limitation dictates treatment with either high doses or more frequent doses, both of which may increase the likelihood of undesirable side effects. To address the need for additional methods to improve the blood half-life of drugs and molecular imaging agents, we developed an "add-on" molecule that contains 3 groups: a truncated Evans blue dye molecule that binds to albumin with a low micromolar affinity and provides a prolonged half-life in the blood; a metal chelate that allows radiolabeling for imaging and radiotherapy; and maleimide for easy conjugation to drug molecules. Methods: The truncated Evans blue molecule was conjugated with the chelator NOTA or DOTA, and the resulting conjugate was denoted as NMEB or DMEB, respectively. As a proof of concept, we coupled NMEB and DMEB to c(RGDfK), which is a small cyclic arginine-glycine-aspartic acid (RGD) peptide, for targeting integrin αvß3 NMEB and DMEB were radiolabeled with 64Cu and 90Y, respectively, and tested in xenograft models. Results: The resulting radiolabeled conjugates showed a prolonged circulation half-life and enhanced tumor accumulation in integrin αvß3-expressing tumors. Tumor uptake was markedly improved over that with NOTA- or DOTA-conjugated c(RGDfK). Tumor radiotherapy experiments in mice with 90Y-DMEB-RGD showed promising results; existing tumors were eliminated. Conclusion: Conjugation of our novel add-on molecule, NMEB or DMEB, to potential tracers or therapeutic agents improved blood half-life and tumor uptake and could transform such agents into theranostic entities.

Perilesional Inflammation in Neurocysticercosis - Relationship Between Contrast-Enhanced Magnetic Resonance Imaging, Evans Blue Staining and Histopathology in the Pig Model.

BACKGROUND: Disease manifestations in neurocysticercosis (NCC) are frequently due to inflammation of degenerating Taenia solium brain cysts. Exacerbated inflammation post anthelmintic treatment is associated with leakage of the blood brain barrier (BBB) using Evans blue (EB) staining. How well EB extravasation into the brain correlates with magnetic resonance imaging (MRI) using gadolinium (Gd) enhancement as a contrast agent and pericystic inflammation was analyzed in pigs harboring brain cysts of Taenia solium. METHODOLOGY/PRINCIPAL FINDINGS: Three groups of 4 naturally infected pigs were assessed. The first and second groups were treated with both praziquantel plus albendazole and sacrificed two and five days post treatment, respectively. A third untreated group remained untreated. Pigs were injected with EB two hours prior to evaluation by Gd-enhanced T1-MRI, and euthanized. The EB staining for each cyst capsule was scored (EB grades were 0: 0%; 1: up to 50%; 2: over 50% but less than 100%; 3: 100%). Similarly, the Gd enhancement around each cyst was qualitatively and quantitatively scored from the MRI. The extent of pericystic inflammation on histology was scored in increasing severity as IS1, IS2, IS3 and IS4. Grade 3 EB staining and enhancement was only seen in treated capsules. Also, treated groups had higher Gd intensity than the untreated group. Grades of enhancement correlated significantly with Gd enhancement intensity. EB staining was correlated with Gd enhancement intensity and with IS4 in the treated groups. These correlations were stronger in internally located cysts compared to superficial cysts in treated groups. SIGNIFICANCE: EB staining and Gd enhancement strongly correlate. The intensity of enhancement determined by MRI is a good indication of the degree of inflammation. Similarly, EB staining highly correlates with the degree of inflammation and may be applied to study inflammation in the pig model of NCC.

Bloodstream-To-Eye Infections Are Facilitated by Outer Blood-Retinal Barrier Dysfunction.

The blood-retinal barrier (BRB) functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE) cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE), a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3) was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu) of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB permeability is not required for the development of EBE, but toxin production may facilitate EBE pathogenesis.

Enhancement of Small Molecule Delivery by Pulsed High-Intensity Focused Ultrasound: A Parameter Exploration.

Chemotherapeutic drug delivery is often ineffective within solid tumors, but increasing the drug dose would result in systemic toxicity. The use of high-intensity focused ultrasound (HIFU) has the potential to enhance penetration of small molecules. However, operation parameters need to be optimized before the use of chemotherapeutic drugs in vivo and translation to clinical trials. In this study, the effects of pulsed HIFU (pHIFU) parameters (spatial-average pulse-average intensity, duty factor and pulse repetition frequency) on the penetration as well as content of small molecules were evaluated in ex vivo porcine kidneys. Specific HIFU parameters resulted in more than 40 times greater Evans blue content and 3.5 times the penetration depth compared with untreated samples. When selected parameters were applied to porcine kidneys in vivo, a 2.3-fold increase in concentration was obtained after a 2-min exposure to pHIFU. Pulsed HIFU has been found to be an effective modality to enhance both the concentration and penetration depth of small molecules in tissue using the optimized HIFU parameters. Although, performed in normal tissue, this study has the promise of translation into tumor tissue.

Stable Evans Blue Derived Exendin-4 Peptide for Type 2 Diabetes Treatment.

In the treatment of type 2 diabetes mellitus, it is very important to develop therapeutics with prolonged circulation half-life. Exendin-4 is a glucagon like peptide-1 receptor (GLP-1R) agonist that has been modified in different ways for imaging insulinoma and for treating type-2 diabetes. In this work, we synthesized a maleimide derivative of truncated Evans blue dye (MEB-C3-Mal) to conjugate with (Cys(40))exendin-4 to obtain a highly stable MEB-C3-(Cys(40))exendin-4 (denoted as Abextide II). Through in situ binding with endogenous albumin, Abextide II lowers blood glucose level and prolongs the hypoglycemic effect in a type 2 diabetes mouse model more than the FDA approved Albiglutide.

Quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

Dye-sensitized solar cell based on spray deposited ZnO thin film: performance analysis through DFT approach.

A dye-sensitized solar cell based on a spray deposited zinc oxide (ZnO) photoanode with Evans blue as a sensitizer was fabricated. Structural analysis confirms the hexagonal wurtzite phase of the ZnO photoanode with c-axis orientation. Surface morphology of the ZnO photoanode shows uniform distribution of spherically-shaped grains, ranging from 18 nm to 25 nm. The power conversion efficiency of the device was measured as 0.1%. Density functional theory was adopted to study the observed photovoltaic performance of the fabricated device. The analysis of the electronic properties of Evans blue dye showed that it has a pronounced effect on the observed device performance.

Interfacial polymerization for colorimetric labeling of protein expression in cells.

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

Low dose cranial irradiation-induced cerebrovascular damage is reversible in mice.

BACKGROUND: High-dose radiation-induced blood-brain barrier breakdown contributes to acute radiation toxicity syndrome and delayed brain injury, but there are few data on the effects of low dose cranial irradiation. Our goal was to measure blood-brain barrier changes after low (0.1 Gy), moderate (2 Gy) and high (10 Gy) dose irradiation under in vivo and in vitro conditions. METHODOLOGY: Cranial irradiation was performed on 10-day-old and 10-week-old mice. Blood-brain barrier permeability for Evans blue, body weight and number of peripheral mononuclear and circulating endothelial progenitor cells were evaluated 1, 4 and 26 weeks postirradiation. Barrier properties of primary mouse brain endothelial cells co-cultured with glial cells were determined by measurement of resistance and permeability for marker molecules and staining for interendothelial junctions. Endothelial senescence was determined by senescence associated ß-galactosidase staining. PRINCIPLE FINDINGS: Extravasation of Evans blue increased in cerebrum and cerebellum in adult mice 1 week and in infant mice 4 weeks postirradiation at all treatment doses. Head irradiation with 10 Gy decreased body weight. The number of circulating endothelial progenitor cells in blood was decreased 1 day after irradiation with 0.1 and 2 Gy. Increase in the permeability of cultured brain endothelial monolayers for fluorescein and albumin was time- and radiation dose dependent and accompanied by changes in junctional immunostaining for claudin-5, ZO-1 and ß-catenin. The number of cultured brain endothelial and glial cells decreased from third day of postirradiation and senescence in endothelial cells increased at 2 and 10 Gy. CONCLUSION: Not only high but low and moderate doses of cranial irradiation increase permeability of cerebral vessels in mice, but this effect is reversible by 6 months. In-vitro experiments suggest that irradiation changes junctional morphology, decreases cell number and causes senescence in brain endothelial cells.