A simple method for producing multiple copies of controlled release small molecule microarrays for cell-based screening.

Traditional drug discovery involves the screening of lead compounds from a chemical library by using cell-based high throughput screening (HTS) procedures. This has created a demand for the development of cell-based microarray chips for HTS of compounds. Although several cell-based microarray devices and procedures for screening of chemical libraries have been reported, each has limitations in terms of simplicity, speed, and cost. Here, we sought to make a simple method for producing multiple copies of microarray chips for the controlled release of small molecules during cell-based screening. Arrays of polytetrafluoroethylene microchannels were set in poly(dimethylsiloxane) and were formed in a metal mold. Subsequently, a biodegradable polymer, PLGAs, with chemical compounds was injected into each channel, and the array was sliced perpendicular to the channels to create multiple copies of the microarray chip. After seeding the cells on the microarray chip, we were able to successfully control the diffusion of small molecules and locally introduce the compounds into cells. The described method enables the production of multiple copies of the chip by using an easy, rapid, and inexpensive array fabrication without any specialized devices. Moreover, screening using the microarray chip minimizes the consumption of cells and chemicals. Both the biodegradable material and compound injected into each channel can be individually tuned for optimized performance. Therefore, we expect that this method will be useful for developing cell-based HTS assays for small chemical compounds to find drug candidates.

Mechanism of Mitf inhibition and morphological differentiation effects of hirsein A on B16 melanoma cells revealed by DNA microarray.

BACKGROUND: We have previously reported that hirsein A inhibits melanogenesis in B16 melanoma cells by downregulating the Mitf gene expression. OBJECTIVE: In this study, microarray was employed to determine the transcriptional response of B16 cells to hirsein A (HA) treatment and to find out the mechanism underlying Mitf downregulation. METHODS: DNA microarray, spotted with 265 genes for melanogenesis and signal transduction, was performed using the total RNA isolated from B16 cells treated with HA. Validation of the results was done using real-time PCR. In addition, real-time PCR using primers for Mda-7 gene and F-actin staining were performed. Transfection experiments were performed to knockdown the expression of the Mc1r gene to evaluate its role in the cell morphological change observed. RESULTS: As expected, the expressions of the Mitf-regulated melanosome transport genes and the Mc1r gene were downregulated. Furthermore, the expressions of the MAPK pathway intermediates were either up- or downregulated. Genes associated with cell differentiation, such as Gadd45b, were upregulated and prompted us to determine the expression of the Il-24 (Mda-7) gene using real-time PCR. There was an increase in the Mda-7 mRNA expression in B16 and HMV-II melanoma cells, and in human melanocytes. To better visualize the cell morphology, F-actin staining was performed and the results showed an increase in the dendrite outgrowth in HA-treated cells. Silencing the Mc1r gene did not cause a change in the B16 cell morphology observed in cells treated with HA. CONCLUSION: This study demonstrated that HA downregulates Mitf gene expression by regulating the expressions of the MAPK signaling pathway intermediates. In addition, the inhibited Mc1r gene expression also contributed to the overall Mitf downregulation but does not play a role in the observed change in B16 cell morphology. HA surprisingly can regulate genes associated with differentiating cells (Mda-7) suggesting a role for HA in the melanoma cell differentiation induction. While the exact molecular mechanism by which HA promotes cell differentiation remain to be determined, it is clear that HA can downregulate Mitf expression and promote cell differentiation and has the potential to be used in the development of therapy for melanoma.

Development of polymeric drug delivery system for recognizing vascular endothelial dysfunction.

The vascular endothelium plays an important role in regulating vascular homeostasis. Damage to the endothelium can lead to cardiovascular diseases such as arteriosclerosis. Therefore, early-stage detection and evaluation of vascular endothelium dysfunction would be very important for effective diagnosis and therapy. We synthesized a polymeric drug carrier bearing an Evans blue analogue as a probing unit for endothelium injury. The polymeric carrier spontaneously formed stable nanoparticles with micelle-like structure in aqueous media and could encapsulate hydrophobic doxorubicin (DOX). The encapsulated DOX showed a sustainable release profile over a period of 10-60 h depending on the loaded DOX concentration. The polymeric carrier specifically adsorbed against the endothelium-injured site in extracted porcine aorta. These properties of the polymeric drug carrier will be suitable for specific drug delivery to endothelium dysfunctional region.

In vivo magnetic resonance imaging of atherosclerotic lesions with a newly developed Evans blue-DTPA-gadolinium contrast medium in apolipoprotein-E-deficient mice.

BACKGROUND: Magnetic resonance imaging (MRI) contrast agents that specifically detect atherosclerotic plaque may be useful for the noninvasive detection of the plaque. We have recently developed a new contrast agent, Evans blue-DTPA-gadolinium (EB-DTPA-Gd), which selectively accumulates vascular lesions with endothelial removal. In this study, we examined whether EB-DTPA-Gd is also useful for in vivo imaging of atherosclerotic plaques. METHODS: We used male apolipoprotein-E-deficient (ApoE-/-) mice of different ages (3, 6 and 12 months old) and age-matched male wild-type mice. After a single intravenous administration of EB-DTPA-Gd (160 microM/kg body weight), MRI T(1) signal was obtained in vivo. RESULTS: Increased signal intensity in the aortic wall was noted within 10-20 min after intravenous injection of EB-DTPA-Gd and was maintained for 30 min. The MRI enhancement in the aorta of ApoE-/- mice was increased in accordance with age, whereas no such enhancement was noted in wild-type mice. Histological examination demonstrated that there was a topological correlation between the site of MRI enhancement and that of atherosclerotic plaque. CONCLUSIONS: These results indicate that EB-DTPA-Gd is a useful MRI contrast medium for the in vivo detection of atherosclerotic plaques.

In vivo MR detection of vascular endothelial injury using a new class of MRI contrast agent.

A new class of dye-based MRI contrast agents, EB-DTPA-Gd, was designed and synthesized. The contrast agent was found to accumulate at the site of endothelial injury when the reagent was applied to isolated porcine blood vessels or in an ex vivo experiment using rat. In vivo MR detection of vascular endothelial injury was also successful in rat with its common carotid artery injured by balloon treatment. These results indicate that EB-DTPA-Gd is potentially useful for the diagnosis of vascular diseases.

First functionalized MRI contrast agent recognizing vascular lesions.

A new MRI-contrast agent, EB-DTPA-Gd, that has an Evans Blue analogue as a sensing unit for endothelium lesions, was designed and synthesized. The agent also has diethylenetriamine-N,N,N',N",N"-pentaacetic acid-Gd complex (Gadolinium-DTPA) units, which have been used as detection units for T1-weighted MRI. The EB-DTPA-Gd was able to recognize and adsorb to the vascular endothelium-denuded region of porcine aorta, and to decrease the relaxation time of circumferential water's protons, making possible MR imaging of the endothelium-denuded region. The compound can be employed as a contrast agent for the imaging of vascular lesions using MRI.