Regulation of the Human Taurine Transporter by Oxidative Stress in Retinal Pigment Epithelial Cells Stably Transformed to Overexpress Aldose Reductase

In diabetes, overexpression of aldose reductase (AR) and consequent glucose-induced impairment of antioxidant defense systems may predispose to oxidative stress and the development of diabetic complications, but the mechanisms are poorly understood. Taurine (2-aminoethanesulfonic acid) functions as an antioxidant, osmolyte, and calcium modulator such that its intracellular depletion could promote cytotoxicity in diabetes. The relationships of oxidative stress and basal AR gene expression to Na+-taurine cotransporter (TT) gene expression, protein abundance, and TT activity were therefore explored in low AR-expressing human retinal pigment epithelial (RPE) 47 cells and RPE 47 cells stably transformed to overexpress AR (RPE 75). Changes in TT gene expression were determined using a 4.6-kb TT promoter-luciferase fusion gene. Compared with RPE 47 cells, in high AR-expressing RPE 75 cells, TT promoter activity was decreased by 46%, which was prevented by an AR inhibitor. TT promoter activity increased up to 900% by prooxidant exposure, which was associated with increased TT peptide abundance and taurine transport. However, induction of TT promoter activity by oxidative stress was attenuated in high AR-expressing cells and partially corrected by AR inhibitor. Finally, exposure of RPE 75 cells to high glucose increased oxidative stress, but down-regulated TT expression. These studies demonstrate for the first time that the TT is regulated by oxidative stress and that overexpression of AR and high glucose impair this response. Abnormal expression of AR may therefore impair antioxidant defense, which may determine tissue susceptibility to chronic diabetic complications. Antioxid. Redox Signal. 7, 1530–1542.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63284/1/ars.2005.7.1530.pd

Design and Function of a Dendrimer-Based Therapeutic Nanodevice Targeted to Tumor Cells Through the Folate Receptor

Purpose . We sought to develop nanoscale drug delivery materials that would allow targeted intracellular delivery while having an imaging capability for tracking uptake of the material. A complex nanodevice was designed and synthesized that targets tumor cells through the folate receptor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41493/1/11095_2004_Article_378868.pd

Optical Fiber-Based In Vivo Quantification of Growth Factor Receptors

[[abstract]]BACKGROUND: Growth factor receptors such as epidermal growth factor receptor 1 and human epidermal growth receptor 2 (HER2) are overexpressed in certain cancer cells. Antibodies against these receptors (eg. cetuximab and transtuzumab [Herceptin]) have shown therapeutic value in cancer treatment. The existing methods for the quantification of these receptors in tumors involve immunohistochemistry or DNA quantification, both in extracted tissue samples. The goal of the study was to evaluate whether an optical fiber-based technique can be used to quantify the expression of multiple growth factor receptors simultaneously. METHODS: The authors examined HER2 expression using the monoclonal antibody trastuzumab as a targeting ligand to test their system. They conjugated trastuzumab to 2 different Alexa Fluor dyes with different excitation and emission wavelengths. Two of the dye conjugates were subsequently injected intravenously into mice bearing HER2-expressing subcutaneous tumors. An optical fiber was then inserted into the tumor through a 30-gauge needle, and using a single laser beam as the excitation source, the fluorescence emitted by the 2 conjugates was identified and quantified by 2-photon optical fiber fluorescence. RESULTS: The 2 conjugates bound to the HER2-expressing tumor competitively in a receptor-specific fashion, but they failed to bind to a similar cell tumor that did not express HER2. The concentration of the conjugate present in the tumor as determined by 2-photon optical fiber fluorescence was shown to serve as an index of the HER2 expression levels

Investigation of tumor cell targeting of a dendrimer nanoparticle using a double-clad optical fiber probe

[[abstract]]Fluorescence quantification in tissues using conventional techniques can be difficult due to the absorption and scattering of light in tissues. Our previous studies have shown that a single-mode optical fiber (SMF)–based, two-photon optical fiber fluorescence (TPOFF) probe could be effective as a minimally invasive, real-time technique for quantifying fluorescence in solid tumors. We report improved results with this technique using a solid, double-clad optical fiber (DCF). The DCF can maintain a high excitation rate by propagating ultrashort laser pulses down an inner single-mode core, while demonstrating improved collection efficiency by using a high–numerical aperture multimode outer core confined with a second clad. We have compared the TPOFF detection efficiency of the DCF versus the SMF with standard solutions of the generation 5 poly(amidoamine) dendrimer (G5) nanoparticles G5-6TAMRA (G5-6T) and G5-6TAMRA-folic acid (G5-6T-FA). The DCF probe showed three- to five-fold increases in the detection efficiency of these conjugates, in comparison to the SMF. We also demonstrate the applicability of the DCF to quantify the targeted uptake of G5-6T-FA in mouse tumors expressing the FA receptor. These results indicate that the TPOFF technique using the DCF probe is an appropriate tool to quantify low nanomolar concentrations of targeted fluorescent probes from deep tissue

Two-photon Fluorescence Measurement of a Targeted Nanodevice Using a Sensitive Double Clad Optical Fiber

[[abstract]]Fluorescence quantification in tissues using conventional techniques is problematic owing to the absorption and scattering of light in the tissues. Whole body fluorescence imaging techniques do not provide accurate quantitative information on the distribution of a fluorescently tagged molecule in tissues. Owing to the limited tissue penetration of light, these methods also lack sensitivity for detection of low concentrations of tissue fluorescence. Previously we have developed a two-photon optical fiber fluorescence (TPOFF) probe as a minimally invasive technique for quantifying fluorescence in solid tumors in live mice in a real-time basis (Thomas et al, Proceedings of the SPIE, 2006, Vol 6095). In those studies we have used a single mode optical fiber (SMF) through which femtosecond laser pulses were delivered into the tumor, which enabled us to measure low micromolar concentrations of targeted fluorescent nanoparticles. It is essential that a more sensitive TPOFF device is developed for quantification of lower levels of a targeted fluorescent agent. Here we demonstrate the biological application of a double-clad optical fiber (DCF) that can keep high excitation rate by propagating ultrashort laser pulses down an inner single mode core, while improving the collection efficiency by using a high-NA multimode outer core confined with a second clad. The DCF does not have a hole which prevents the capillary suction of biological fluids which is a problem for the biological application of a previously described Double Clad Photonic Crystal Fiber (DCPCF, Ye, et al, Proceedings of the SPIE, 2005. 5700: p. 23-27). The solid DCF used has a numeric aperture of 0.46, which is smaller than that of a DCPCF. Although it does not achieve as high a collection efficiency as the DCPCF, it provides significant improvement over traditional single-clad fibers. We have compared the two-photon fluorescence detection efficiency of using the DCF vs. SMF with standard solutions of the dye 6-TAMRA (6T), and the generation 5 dendrimer (G5) nanoparticles G5-6T and G5-6T-Folic acid (G5-6T-FA). We have observed about 6-fold increase in the detection efficiency of these fluorescent agents. We then compared the targeting of G5-6T-FA in FA receptor (FAR)-expressing cells in vitro, and finally demonstrated the applicability of the DCF fiber to quantify the in vivo targeted uptake of G5-6T-FA in mice tumor expressing FAR. In summary, the DCF-TPOFF probe is an appropriate tool to quantify nanomolar levels of a targeted nanoparticle in deep tissue in vivo

The DCCT, metabolism and diabetic neuropathy: perspectives for the fourth international symposium on diabetic neuropathy

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35015/1/201_ftp.pd

PSMA-Targeted Stably Linked “Dendrimer-Glutamate Urea-Methotrexate” as a Prostate Cancer Therapeutic

One of the important criteria for achieving efficient nanoparticle-based targeted drug delivery is that the drug is not prematurely released at off-target sites. Here we report the preclinical evaluation of a serum-stable dendrimer-based drug conjugate capable of actively targeting into prostate cancer (PC) cells, delivered through the prostate-specific membrane antigen (PSMA). Multiple molecules of PSMA-binding small molecule glutamate urea (GLA; targeting agent) and the drug methotrexate (MTX) were conjugated to generation 5 PAMAM dendrimer (G5) through Cu-free “click” chemistry. The GLA was conjugated through a stable amide bond, and the MTX was conjugated either through ester (Es)- or amide (Am)-coupling, to generate G5-GLA_<i>m</i>-(Es)­MTX_<i>n</i> and G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i>, respectively. In serum-containing medium, free MTX was slowly released from “G5-GLA_<i>m</i>-(Es)­MTX_<i>n</i>”, with ∼8% MTX released from the dendrimer in 72 h, whereas the MTX on G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i> was completely stable. The G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i> bound and internalized into PSMA-expressing LNCaP cells, but not into PSMA-negative PC3 cells. The conjugate-inhibited recombinant dihydrofolate reductase and induced potent cytotoxicity in the LNCaP cells, but not in the PC3 cells. Similar to the action of free GLA, stable amide-linked dendrimer-GLA was capable of inhibiting the enzyme N-acetylated α-linked acidic dipeptidase (NAALADase) activity of PSMA. The G5-GLA_<i>m</i>-MTX_<i>n</i> may serve as a serum-stable nanoparticle conjugate to specifically and effectively target and treat PSMA-overexpressing prostate tumors