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Supplementary Figures 1-8 from Sustained Small Interfering RNA Delivery by Mesoporous Silicon Particles

Cerebrovascular changes occur in Alzheimer’s disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson’s disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.

High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune‐modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin–nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα‐CSG fusion protein to tumour ECM in tumour‐bearing mice. Intravenously injected TNFα‐CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM‐rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour‐bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα., This study establishes a new approach to treat solid tumours by immune modulation and ECM depletion. The developed agent, TNFα‐CSG, has dual capacity as an immunotherapeutic and ECM reducing agent which improves tumour perfusion and drug delivery.

© 2016 The Royal Society of Chemistry. We report the synthesis, characterization, and assessment of a nanoparticle-based RNAi delivery platform that protects siRNA payloads against nuclease-induced degradation and efficiently delivers them to target cells. The nanocarrier is based on biodegradable mesoporous silicon nanoparticles (pSiNPs), where the voids of the nanoparticles are loaded with siRNA and the nanoparticles are encapsulated with graphene oxide nanosheets (GO-pSiNPs). The graphene oxide encapsulant delays release of the oligonucleotide payloads in vitro by a factor of 3. When conjugated to a targeting peptide derived from the rabies virus glycoprotein (RVG), the nanoparticles show 2-fold greater cellular uptake and gene silencing. Intravenous administration of the nanoparticles into brain-injured mice results in substantial accumulation specifically at the site of injury.

Cerebrovascular changes occur in Alzheimer’s disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson’s disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation., Cerebrovascular changes and astrogliosis occur in Alzheimer’s disease (AD). Using an in vivo phage display technique, the authors identified a peptide that upon systematic administration, can home to brain endothelial cells and astrocytes in mouse models of AD at the early stages of the disease.

Bacterial resistance to antibiotics has made it necessary to resort to using antibacterial drugs that have considerable toxicities. Here, we show that conjugation of vancomycin-loaded nanoparticles with the cyclic 9-amino-acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of the nanoparticles in S. aureus-infected tissues and reduces the systemic dose needed, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing CARG in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissues may help combat difficult-to-treat infections. Nanoparticles carrying an antibiotic and conjugated with a peptide identified via phage display that binds specifically to Staphylococcus aureus effectively suppress staphylococcal infections in vivo.

Anti-angiogenic and vascular disrupting therapies rely on the dependence of tumors on new blood vessels to sustain tumor growth. We previously reported a potent vascular disrupting agent, a theranostic nanosystem consisting of a tumor vasculature-homing peptide (CGKRK) fused to a pro-apoptotic peptide [D(KLAKLAK)2] coated on iron oxide nanoparticles. This nanosystem showed promising therapeutic efficacy in glioblastoma (GBM) and breast cancer models. However, complete control of the tumors was not achieved, and some tumors became non-responsive to the treatment. Here we examined the non-responder phenomenon in an aggressive MCF10-CA1a breast tumor model. In the treatment-resistant tumors we noted the emergence of CD31-negative patent neovessels and a concomitant loss of tumor homing of the nanosystem. In vivo phage library screening in mice bearing non-responder tumors showed that compared to untreated and treatment-sensitive tumors, treatment sensitive tumors yield a distinct landscape of vascular homing peptides characterized by over-representation of peptides that target αv integrins. Our approach may be generally applicable to the development of targeted therapies for tumors that have failed treatment.

Traumatic brain injury (TBI) is a major health and socio-economic problem, but no pharmacological agent is currently approved for the treatment of acute TBI. Thus, there is a great need for advances in this field. Here, we describe a short peptide (sequence CAQK) identified by in vivo phage display screening in mice with acute brain injury. The CAQK peptide selectively binds to injured mouse and human brain, and systemically injected CAQK specifically homes to sites of brain injury in mouse models. The CAQK target is a proteoglycan complex upregulated in brain injuries. Coupling to CAQK increased injury site accumulation of systemically administered molecules ranging from a drug-sized molecule to nanoparticles. CAQK-coated nanoparticles containing silencing oligonucleotides provided the first evidence of gene silencing in injured brain parenchyma by systemically administered siRNA. These findings present an effective targeting strategy for the delivery of therapeutics in clinical management of acute brain injuries., Accurate treatment of traumatic brain injuries, a leading cause of neurological disability and death in young people, is hampered by poor accumulation of drugs in the brain. Here, the authors describe a tetrapeptide that can efficiently target brain injuries and deliver therapeutic or diagnostic payload.

CD44, the primary receptor for hyaluronic acid, plays an important role in tumor growth and metastasis. CD44-hyaluronic acid interactions can be exploited for targeted delivery of anticancer agents specifically to cancer cells. Although various splicing variants of CD44 are expressed on the plasma membrane of cancer cells, the hyaluronic acid binding domain (HABD) is highly conserved among the CD44 splicing variants. Using a novel two-step process, we have identified monothiophosphate-modified aptamers (thioaptamers) that specifically bind to the CD44's HABD with high affinities. Binding affinities of the selected thioaptamers for the HABD were in the range of 180-295 nM, an affinity significantly higher than that of hyaluronic acid (K(d) above the micromolar range). The selected thioaptamers bound to CD44 positive human ovarian cancer cell lines (SKOV3, IGROV, and A2780) but failed to bind the CD44 negative NIH3T3 cell line. Our results indicated that thio substitution at specific positions of the DNA phosphate backbone results in specific and high-affinity binding of thioaptamers to CD44. The selected thioaptamers will be of great interest for further development as a targeting or imaging agent for the delivery of therapeutic payloads for cancer tissues.

Aman P. Mann 1,6 , Rohan C. Bhavane 2 , Anoma Somasunderam 3 , Brenda Liz Montalvo-Ortiz 4 , Ketan B. Ghaghada 2 , David Volk 3 , Rene Nieves-Alicea 5 , K. Stephen Suh 5 , Mauro Ferrari 6 , Ananth Annapragada 2 , David G. Gorenstein 3 , Takemi Tanaka 4 1 Department of Nanomedicine, University of Texas Health Science Center at Houston, 1825 Hermann Pressler, Houston, Texas, 77030 2 School of Biomedical Informatics, University of Texas Health Science Center at Houston, 7000 Fannin, Houston, Texas, 77030 3 Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 1825 Hermann Pressler, Houston, Texas, 77030 4 Department of Pharmaceutical Sciences, Thomas Jefferson University,130 South 9th Street, Philadelphia, PA, 19107 5 The John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, New Jersey, 07601 6 The Methodist Hospital Research Institute, Houston, Texas, 77030 Keywords: drug delivery; aptamer; E-selectin Received: April 11, 2011; Accepted: May 6, 2011; Published: May 7, 2011; Correspondence: Takemi Tanaka, e-mail: // // Abstract 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.

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)–/CD44+ hormone-independent breast cancer cells, but not of the ER+/CD44-/low hormone-dependent breast cancer cells. Coincidentally, CD44+ breast cancer cells were abundant in metastatic lung and brain lesions in ER– breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER–/CD44+ breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44+ cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER–/CD44+ breast cancer.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

RNA interference (RNAi) is a powerful approach for silencing oncogenes; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained delivery. Here, we describe a novel approach to overcome these limitations using mesoporous silicon particles loaded with nanoparticles (i.e., liposomes) containing small interfering RNA (siRNA) targeted against oncoprotein that contributes to cancer cell survival. This delivery method resulted in sustained gene silencing for at least 3 weeks with substantial reduction of tumor growth with no overt toxicities in two independent orthotopic mouse models of ovarian cancer following a single intravenous administration of mesoporous silicon particles loaded with liposomal EphA2-siRNA.

The involvement of E-selectin in cancer has long been recognized based on histopathological studies, and the important role of E-selectin in cancer progression and metastasis was further reinforced by a number of basic researches. Both the solid state and soluble forms of E-selectin have been tested as a possible biomarker for different cancer types for detection as well as for monitoring. While a number of studies have suggested the potential usefulness of E-selectin as a biomarker, the results from clinical trials are somewhat controversial and it has not been translated into clinical utility. It appears that the use of E-selectin as a single biomarker for diagnosis and prognosis may require more careful evaluation. The utility of E-selectin as a biomarker in conjunction with other biomarkers that are relevant to the biological cascade for E-selectin or are critical for patient prognosis may open up a new venue to further establish E-selectin as a cancer biomarker.

Cancer is a leading cause of morbidity and mortality worldwide, with recent advancements resulting in modest impacts on patient survival. Nanomedicine represents an innovative field with immense potential for improving cancer treatment, having ushered in several established drug delivery platforms. Nanoconstructs such as liposomes are widely used in clinics, while polymer micelles are in advanced phases of clinical trials in several countries. Currently, the field of nanomedicine is generating a new wave of nanoscale drug delivery strategies, embracing trends that involve the functionalization of these constructs with moieties that enhance site-specific delivery and tailored release. Herein, we discuss several advancements in established nanoparticle technologies such as liposomes, polymer micelles, and dendrimers regarding tumor targeting and controlled release strategies, which are being incorporated into their design with the hope of generating a more robust and efficacious nanotherapeutic modality. We also highlight a novel strategy known as multistage drug delivery; a rationally designed nanocarrier aimed at overcoming numerous biological barriers involved in drug delivery through the decoupling of various tasks that comprise the journey from the moment of systemic administration to arrival at the tumor site.

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. Cancer Res; 70(9); 3687–96. ©2010 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases and is virtually incurable. The molecular mechanisms that contribute to the intrinsic resistance of PDAC to various anticancer therapies are not well understood. Recently, we have observed that several drug-resistant and metastatic tumors and tumor cell lines expressed elevated levels of tissue transglutaminase (TG2). Because PDAC exhibits inherent resistance to various drugs, we determined the constitutive expression of TG2 in 75 PDAC and 12 PDAC cell lines. Our results showed that 42 of 75 (56%) PDAC tumor samples expressed higher basal levels of TG2 compared with the normal pancreatic ducts [odds ratio (OR), 2.439; P = 0.012]. The increased expression of TG2 in PDAC was strongly associated with nodal metastasis (OR, 3.400; P = 0.017) and lymphovascular invasion (OR, 3.055; P = 0.045). Increased expression of TG2 was also evident in all 12 cell lines examined. The elevated expression of TG2 in PDAC cell lines was associated with gemcitabine resistance and increased invasive potential. Overexpression of catalytically active or inactive (C277S mutant) TG2 induced focal adhesion kinase (FAK) activation and augmented invasive functions in the BxPC-3 cell line. Conversely, down-regulation of TG2 by small interfering RNA attenuated FAK phosphorylation. Immunoprecipitation and confocal microscopy data revealed that TG2 was associated with FAK protein in PDAC cells. The activated FAK colocalized with TG2 at focal adhesion points. These results show for the first time that elevated expression of TG2 can induce constitutive activation of FAK and thus may contribute to the development of drug resistance and invasive phenotypes in PDAC. (Cancer Res 2006; 66(21): 10525-33)

The transcription factor nuclear factor-κB (NF-κB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-κB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-κB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-κB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-κB. Activation of TG2 led to activation of NF-κB; conversely, inhibition of TG2 activity inhibited activation of NF-κB. Similarly, ectopic expression of TG2 caused activation of NF-κB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-κB. Our results further indicated that constitutive NF-κB reporter activity in pancreatic cancer cells is not affected by dominant-negative IκBα. Additionally, coimmunoprecipitation and confocal microscopy showed that IκBα is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-κB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-κB in tumor cells via a novel pathway that is most likely independent of IκBα kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-κB activation and rendering cancer cells sensitive to anticancer therapies. (Cancer Res 2006; 66(17): 8788-95)

Breast cancer patients' outcome and survival depends on the timely diagnosis of early malignant lesions. The lack of molecular understanding of the early changes in breast tissue that facilitate tumor progression has limited the development of tools to non-invasively distinguish early stage disease from normal breast tissue. Therefore, probes to target and better understand early breast tumors are much needed. Our laboratory has pioneered in vivo screening of phage libraries to identify peptides that specifically recognize tumor vessels, including breast cancer vasculature. These peptides have been employed to specifically deliver drugs, diagnostic agents, and nanoparticles to breast tumors. Breast cancer progression constitutes a multistep process through a series of intermediate hyperplastic and neoplastic stages to invasive carcinoma. In this study, we aimed to identify peptides that specifically recognize premalignant lesions in the mammary tissue. To achieve this goal, we utilized the power of phage display to probe hyperplastic lesions associated with premalignant disease in a transgenic MMTV-PyMT animal model. After multiple ex-vivo and in-vivo rounds of selection, we identified a peptide, Prem-1, that on intravenous administration, specifically homed to premalignant mammary lesions. Prem-1 also homed to fully developed breast tumors in the same animal model, suggesting that the putative receptor for Prem-1 is expressed throughout the progression of the disease. Interestingly, Prem-1 did not show any affinity to normal breast tissue. Furthermore, we also identified 2 other candidate peptides that showed significant homing to premalignant lesions with a very different binding pattern as compared to Prem-1. We hypothesized that all three peptides recognize early changes in the breast tissue microenvironment but each bind a different target receptor in the tissue. We are currently investigating these receptors and analyzing their expression in breast cancer progression. Secondly, we are testing the peptides identified herein for the delivery of therapeutic nanoparticles as a mode of early intervention in breast cancer progression. This project utilized the natural environment in the early breast tumor to probe for new markers for detection of early disease. Methods to detect and study early premalignant mammary lesions will expand our understanding of the natural history of the disease, especially the changes in breast tissue that likely lead to tumor development. Hence, the knowledge gained from this study would provide a basis for therapies aimed at suppressing or eradicating premalignant breast lesions. This would be particularly beneficial for women at high-risk of breast cancer based on their genetic predisposition (mutations in BRCA genes). Citation Format: Aman P. Mann, Ramana Kotamraju, Tambet Teesalu, Erkki Ruoslahti. Targeting premalignant lesions for early breast cancer detection and intervention. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3258. doi:10.1158/1538-7445.AM2014-3258

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 (K(D) = 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 sLe(x) 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.

E-selectin (CD62E, ELAM-1 or LECAM-2) is a calcium dependent cell surface glycoprotein that mediates an adhesion of leukocytes on inflamed endothelium through recognition of specific carbohydrate ligands, sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA). E-selectin expression is transcriptionally induced by NF-κB and AP-1 in response to inflammatory cytokines such as IL-1 and TNF-alpha. Accordingly, elevated E-selectin expression was reported in many types of acute and chronic inflammatory diseases including cancer. Aberrant homing of leukocytes to tumor results in tumor progression and local invasion. In addition, E-selectin and its counter ligands have also been reported to trigger the initial adhesive step of circulating cancer cells to endothelial cells. Therefore, blockade of E-selectin is a promising therapeutic strategy to control pathological infiltration of leukocytes and/or metastatic cancer cells. Thioaptamers (TA) are single stranded DNA molecules with thio substitution in the phosphate backbone, which provide stability against nucleases. Thioaptamer can fold into tertiary structures and bind with high affinity and specificity to a target molecule, and thus suitable for in vivo applications. In this study, we aimed to identify thioaptamer ligands that bind to E-selectin with high affinity and specificity. To achieve this goal, we used two-step selection strategies, recombinant protein based thioaptamer selection from a combinatorial thioaptamer library that contains 10E14 random sequences, followed by a cell based binding to tetracycline inducible E-selectin endothelial cells. We selected 14 different groups of thioaptamer candidates following 10 iterative cycles of binding to recombinant E-selectin protein. Using Tet-on inducible E-selectin endothelial cells, we identified the lead three TA candidates that bind to E-selectin on the surface of endothelial cells at high affinity (nM range) and specificity. In addition, the TA bound to tumor associated vasculature that express E-selectin. Moreover, the TA selected demonstrated significant inhibition of leukocyte adhesion to E-selectin positive endothelial cells at low nanomolar concentrations. In summary, we identified antagonistic TA against E-selectin that binds to endothelial cells and interferes with leukocyte bindings at nM range. Potential applications of this TA could be for 1) inflamed tumor vasculature selective targeted delivery of therapeutic molecules or drug delivery carriers 2) the prevention of pathological diapedesis of leukocytes and/or metastatic cancer cells. We are currently investigating potential clinical applications of the TA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5712.