Identification of Thioaptamer Ligand against E-Selectin: Potential Application for Inflamed Vasculature Targeting

Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA) against E-selectin (ESTA-1) by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (KD = 47 nM) while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition) of sLex positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1) that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery

Thioaptamer Conjugated Liposomes for Tumor Vasculature Targeting

Recent developments in multi-functional nanoparticles offer a great potential for targeted delivery of therapeutic compounds and imaging contrast agents to specific cell types, in turn, enhancing therapeutic effect and minimizing side effects. Despite the promise, site specific delivery carriers have not been translated into clinical reality. In this study, we have developed long circulating liposomes with the outer surface decorated with thioated oligonucleotide aptamer (thioaptamer) against E-selectin (ESTA) and evaluated the targeting efficacy and PK parameters. In vitro targeting studies using Human Umbilical Cord Vein Endothelial Cell (HUVEC) demonstrated efficient and rapid uptake of the ESTA conjugated liposomes (ESTA-lip). In vivo, the intravenous administration of ESTA-lip resulted in their accumulation at the tumor vasculature of breast tumor xenografts without shortening the circulation half-life. The study presented here represents an exemplary use of thioaptamer for targeting and opens the door to testing various combinations of thioaptamer and nanocarriers that can be constructed to target multiple cancer types and tumor components for delivery of both therapeutics and imaging agents

Expression of glutathione S-transferase π and α isoforms in breast cancer cell lines

Expression of glutathione S-transferase (GST)-π and GST-α isoforms in breast cancer cell lines. Protein lysates were obtained from exponentially growing MDA-MB-231, F10, and MCF-7/COX-2 cells. Western blot analyses using polyclonal GST-π and GST-α antibodies were performed. β-actin was used as a loading control.<p><b>Copyright information:</b></p><p>Taken from "TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/3/R44</p><p>Breast Cancer Research : BCR 2008;10(3):R44-R44.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2481491.</p><p></p

JS-K, but not JS-43-126, decreases the invasiveness of breast cancer cells across Matrigel

MDA-MB-231, F10, and MCF-7/COX-2 cells were added into Matrigel-coated transwell inserts and incubated with JS-K or JS-43-126 for 72 hours. Cells that invaded through the pores onto the lower side of the filter were fixed, stained, and photographed. The number of invaded cells for each filter was counted in five fields. The invasiveness of the cells is expressed as the mean number of cells that invaded to the lower side of the filter. Columns indicate the mean of triplicate wells ± standard deviation. *Significant decrease in the number of invaded cells relative to untreated cells, < 0.05. MDA-MB-231, F10, and MCF-7/COX-2 cells were plated on Matrigel-coated wells and incubated with JS-K. After 72 hours, cell proliferation was determined by Celltiter 96AQnonradioactive cell proliferation assay. Columns indicate the mean of pentaplicate wells ± standard deviation. MDA-MB-231 and F10 cells were added into type I collagen-coated transwell inserts and incubated with JS-K for 72 hours. Cells that invaded through the pores onto the lower side of the filter were fixed, stained, and photographed.<p><b>Copyright information:</b></p><p>Taken from "TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/3/R44</p><p>Breast Cancer Research : BCR 2008;10(3):R44-R44.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2481491.</p><p></p

JS-K, but not JS-43-126, increases nitric oxide production in breast cancer cells

Conditioned medium supernatant was collected from MDA-MB-231, F10, and MCF-7/COX-2 cells treated in the absence or presence of JS-K or JS-43-126 for 72 hours. Total nitric oxide (NO) was determined by quantifying nitrite, the stable end product of NO oxidation, spectrophotometrically using a colorimetric nonenzymatic nitric oxide assay kit. Nitrite values were normalized for total cell counts and expressed as picomoles per 10cells. Columns indicate the mean of triplicate wells ± standard deviation. *Significant increase in NO levels relative to untreated cells, < 0.05.<p><b>Copyright information:</b></p><p>Taken from "TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/3/R44</p><p>Breast Cancer Research : BCR 2008;10(3):R44-R44.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2481491.</p><p></p

Sustained small interfering RNA delivery by mesoporous silicon particles

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms