Glyceryl Tristearate-Based Lipid Microparticles Loaded with the Tattoo Colorant, Acid Red 87: Colorant Retention Capacity in Excised Porcine Skin.

BACKGROUND: With the increasing diffusion of tattooing, the photolability of tattoo inks has become a critical issue, as available data indicated that several tattoo colorants are unstable under sunlight, generating potentially toxic photodegradation products. Therefore, it is desirable to enhance the photostability of coloring agents contained in tattoo inks. AIMS: Lipid microparticles (LMs) highly loaded with Acid Red 87 (C.I. 45380), a colorant used in tattoo inks, were evaluated for their effect on the colorant photoinstability. In addition, the capacity of the LMs to retain the incorporated C.I. 45380 colorant after their intradermal administration in excised porcine skin was investigated. METHODS: LMs loaded with C.I. 45380 were prepared using glyceryl tristearate as the lipidic material and phosphatidylcholine as the surfactant. Non-encapsulated C.I. 45380 or the colorant-loaded LMs were irradiated with a solar simulator for photodecomposition studies or introduced in the excised porcine skin mounted in Franz diffusion cells for stability evaluation in the dermal tissue. RESULTS AND CONCLUSION: The colorant content of the microparticles was 17.7%, and their size ranged from 25 to 170 µm. The light-induced degradation of C.I. 45380 was significantly decreased by its incorporation in the LMs from 20.2 ± 5.8% to 1.9 ± 2.1%. Moreover, after intradermal injection of free or microencapsulated C.I. 45380 in the excised pig skin, the LMs reduced by 93.7% (from 24.6 to 1.5%) the quantity of the colorant diffused and hence lost in the Franz cell receptor fluid. Hence, the LM carrier efficiently retained the entrapped C.I. 45380 following incubation in the dermal region of the isolated porcine skin, which is in favor of a long-lasting tattoo. Based on these data, the incorporation of C.I. 45380 in the LMs could represent a potentially useful strategy to reduce the photodecomposition of the tattoo colorant and its harmful interactions with the skin tissue.

Stain Deconvolution Using Statistical Analysis of Multi-Resolution Stain Colour Representation.

Stain colour estimation is a prominent factor of the analysis pipeline in most of histology image processing algorithms. Providing a reliable and efficient stain colour deconvolution approach is fundamental for robust algorithm. In this paper, we propose a novel method for stain colour deconvolution of histology images. This approach statistically analyses the multi-resolutional representation of the image to separate the independent observations out of the correlated ones. We then estimate the stain mixing matrix using filtered uncorrelated data. We conducted an extensive set of experiments to compare the proposed method to the recent state of the art methods and demonstrate the robustness of this approach using three different datasets of scanned slides, prepared in different labs using different scanners.

Temperature-responsive magnetite/PEO-PPO-PEO block copolymer nanoparticles for controlled drug targeting delivery.

In this study, temperature-responsive magnetite/polymer nanoparticles were developed from iron oxide nanoparticles and poly(ethyleneimine)-modified poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer. The particles were characterized by TEM, XRD, DLS, VSM, FTIR, and TGA. A typical product has an approximately 20 nm magnetite core and an approximately 40 nm hydrodynamic diameter with a narrow size distribution and is superparamagnetic with large saturation magnetization (51.34 emu/g) at room temperature. The most attractive feature of the nanoparticles is their temperature-responsive volume-transition property. DLS results indicated that their average hydrodynamic diameter underwent a sharp decrease from 45 to 25 nm while evaluating the temperature from 20 to 35 degrees C. The temperature-dependent evolution of the C-O stretching band in the FTIR spectra of the aqueous nanoparticles solution revealed that thermo-induced self-assembly of the immobilized block copolymers occurred on the magnetite solid surfaces, which is accompanied by a conformational change from a fully extended state to a highly coiled state of the copolymer. Consequently, the copolymer shell could act as a temperature-controlled "gate" for the transit of guest substance. The uptake and release of both hydrophobic and hydrophilic model drugs were well controlled by switching the transient opening and closing of the polymer shell at different temperatures. A sustained release of about 3 days was achieved in simulated human body conditions. In primary mouse experiments, drug-entrapped magnetic nanoparticles showed good biocompatibility and effective therapy for spinal cord damage. Such intelligent magnetic nanoparticles are attractive candidates for widespread biomedical applications, particularly in controlled drug-targeting delivery.

Quantitative analysis of the interaction between the fluorescent probe eosin and the Na+/K+-ATPase studied through Rb+ occlusion.

We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K(+)-congener Rb(+) in the Na(+)/K(+)-ATPase. Eosin decreases the equilibrium levels of occluded Rb(+), this effect being fully attributable to a decrease in the apparent affinity of the enzyme for Rb(+) since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na(+)/K(+)-ATPase, both to the Rb(+)-free and to the Rb(+)-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb(+) occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that, like ATP, it increases the velocity of deocclusion of Rb(+). Interactions between eosin and ATP on Rb(+)-release experiments seem to indicate that eosin binds to the low-affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.

Nitric oxide is involved in ischemia-induced apoptosis in brain: a study in neuronal nitric oxide synthase null mice.

Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.

Whole-cell patch-clamp: true perforated or spontaneous conventional recordings?

Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.

Anti-drug antibodies as drug carriers. I. For small molecules.

PURPOSE: To better understand the pharmacokinetics of drugs compounds that bind endogenous antibodies METHODS: Three groups of mice with differing anti-fluorescein (FL) titers were established by empirically developed immunization protocols. These with two control groups were given intravenously [3H]-ethanolamine conjugate of FL (FL-EA). The latter was synthesized using isothiocyanate chemistry. Radioactivity in the circulation, and occasionally in peritoneal ascites, was monitored for 7 days. A group of mice was immunized with eosin Y and given FL-EA. Conversely, eosin Y conjugate of radiolabeled EA (EY-EA) was given to mice immunized with FL. These two groups represented animals of low affinity to probe haptens. The affinity was assessed by a precipitation procedure, while titer was determined by a standard ELISA. Dose of FL-EA varied over a 100-fold. RESULTS: On average, the three immunized groups showed a 1:13:85 ratio of anti-FL titer, with remarkably consistent levels within each group. Elimination rates of FL-EA from the serum of very high-titer mice and high-titer mice were similar, however, were substantially lower than that found in low-titer mice. The latter was in turn lower than that found in non- or mock-immunized mice. Serum of mice immunized with FL showed approximately 200-fold lower affinity towards EY-EA than FL-EA. In these mice and in mice immunized with eosin Y and given FL-EA, the elimination of the probe haptens was again fast, reminiscent of low-titer mice. Mice of either low titer or low affinity showed more rapid redistribution of the conjugate between serum and peritoneal fluid. In a group of mice with comparable anti-FL titer, elimination from serum was independent of dose over a 100-fold difference. The bi-phasic concentration-time profile observed was accommodated by a physiologically meaningful pharmacokinetic model incorporating two compartments in which antibody binding can occur. CONCLUSIONS: Monovalent antigenic substance cannot trigger immune clearance. As such, endogenous antibodies that recognize the molecule can serve as a carrier to result in a substantial decrease in clearance.

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability.

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.

Evaluation of barrier creams: an in vitro technique on human skin.

A method was developed to measure in vitro on human skin the effectiveness of barrier creams against three dyes (eosin, methyl-violet and oil red O) with different n-octanol/water partition coefficients (0.19, 29.8 and 165, respectively). Some galenic properties (water washability, water content and viscosity) of the products were also evaluated to try to understand the mechanisms of such a protection. The barrier creams were assayed by measurements of the dyes in the epidermis of protected skin samples after an application time of 30 min. Whereas some products showed some degree of protection, as claimed on the packaging, we demonstrated in several cases disagreement with the manufacturer's information. Surprisingly, petrolatum was found to provide the best protection of all tested products in our in vitro model. There was no correlation between the galenic parameters of the assayed products and the level of protections, indicating that neither the water content nor the consistence of the formulations influenced the protection effectiveness. In conclusion, regarding the possible skin effects of some irritants, our results stress that barrier creams should be used with caution, knowing the protection limits of some of the formulations marketed.

Influence of phenobarbital and triiodothyronine on the development of hepatic eosine transport in rats.

In male Wistar rats aged 20 and 60 days the influence of a 5-day-treatment with phenobarbital (PB, 75 mg/kg b.m. i.p.), triiodothyronine (T3, 100 micrograms/kg b.m. s.c.) and both agents simultaneously (PB+T3) on eosine accumulation of liver slices in vitro and biliary excretion of eosine in vivo was investigated. PB enhanced bile acid independent bile flow. T3 accelerated eosine accumulation in liver slices. The combined treatment with PB+T3 had no additional effects. The simultaneous action of PB+T3 could not induce the maturation of the rate limiting excretion process for organic anions in 20-day-old rats.