Selection of Aptamers against Live Bacterial Cells

Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6−8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 47 aptamer molecules were bound to a target cell with an apparent K_d of 13 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.

Drug Delivery with Carbon Nanotubes for In vivo Cancer Treatment

Zhuang Liu, Kai Chen, Corrine Davis, Sarah Sherlock, Qizhen Cao, Xiaoyuan Chen and Hongjie Dai

Key Words: Carbon nanotubes • paclitaxel • drug delivery • cancer therapy • nanobiotechnology

Chemically functionalized single-walled carbon nanotubes (SWNT) have shown promise in tumor-targeted accumulation in mice and exhibit biocompatibility, excretion, and little toxicity. Here, we show in vivo SWNT drug delivery for tumor suppression in mice. We conjugate paclitaxel (PTX), a widely used cancer chemotherapy drug, to branched polyethylene glycol chains on SWNTs via a cleavable ester bond to obtain a water-soluble SWNT-PTX conjugate. SWNT-PTX affords higher efficacy in suppressing tumor growth than clinical Taxol in a murine 4T1 breast cancer model, owing to prolonged blood circulation and 10-fold higher tumor PTX uptake by SWNT delivery likely through enhanced permeability and retention. Drug molecules carried into the reticuloendothelial system are released from SWNTs and excreted via biliary pathway without causing obvious toxic effects to normal organs. Thus, nanotube drug delivery is promising for high treatment efficacy and minimum side effects for future cancer therapy with low drug doses.

[Cancer Res 2008;68(16):6652–60]

A Stochastic Model for Cancer Stem Cell Origin in Metastatic Colon Cancer

Christine Odoux, Helene Fohrer, Toshitaka Hoppo, Lynda Guzik, Donna Beer Stolz, Dale W. Lewis, Susanne M. Gollin, T. Clark Gamblin, David A. Geller and Eric Lagasse

Key Words: Chromosomal Instability in Cancer Stem Cells

Human cancers have been found to include transformed stem cells that may drive cancer progression to metastasis. Here, we report that metastatic colon cancer contains clonally derived tumor cells with all of the critical properties expected of stem cells, including self-renewal and the ability to differentiate into mature colon cells. Additionally, when injected into mice, these cells initiated tumors that closely resemble human cancer. Karyotype analyses of parental and clonally derived tumor cells expressed many consistent (clonal) along with unique chromosomal aberrations, suggesting the presence of chromosomal instability in the cancer stem cells. Thus, this new model for cancer origin and metastatic progression includes features of both the hierarchical model for cancerous stem cells and the stochastic model, driven by the observation of chromosomal instability. 

[Cancer Res 2008;68(17):6932–41]

Targeting V600EB-Raf and Akt3 Using Nanoliposomal-Small Interfering RNA Inhibits Cutaneous Melanocytic Lesion Development

Melissa A. Tran, Raghavendra Gowda, Arati Sharma1, Eun-Joo Park, James Adair, Mark Kester, Nadine Barrie Smith and Gavin P. Robertson

Key Words: melanoma • ultrasound • B-Raf • nanoliposomes • siRNA

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and 60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation (V600EB-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, 70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting V600EB-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound–mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting V600EB-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting V600EB-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of V600EB-Raf and Akt3 led to a cooperatively acting 65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting V600EB-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas. 

[Cancer Res 2008;68(18):7638–49]

Quantitative Molecular Magnetic Resonance Imaging of Tumor Angiogenesis Using cNGR-Labeled Paramagnetic Quantum Dots

Marlies Oostendorp, Kim Douma, Tilman M. Hackeng, Anouk Dirksen, Mark J. Post, Marc A.M.J. van Zandvoort and Walter H. Backes

 

Key Words: molecular magnetic resonance imaging • tumor angiogenesis • quantum dots • cNGR • two-photon laser scanning microscopy

 

The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor-bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature. 

[Cancer Res 2008;68(18):7676–83]

Carbon nanotubes as photoacoustic molecular imaging agents in living mice

Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques. As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously, but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects.

http://www.nature.com/nnano/journal/v3/n9/abs/nnano.2008.231.html

Fluorescence Lifetime Correlation Spectroscopic Study of Fluorophore-Labeled Silver Nanoparticles

In this paper, we introduce the use of fluorescence lifetime correlation spectroscopy to study the metal−-fluorophore interactions in solution at the single-fluorophore level. A single-stranded oligonucleotide was chemically bound to a 50-nm-diameter single silver particle, and a Cy5-labeled complementary single-stranded oligonucleotide was hybridized with the silver particle-bound oligonucleotide. The distance between the fluorophore and silver particle was maintained by a rigid hybridized DNA duplex of 8 nm in length. The single Cy5-DNA-Ag particles showed more than 10-fold increase in fluorescence intensity and a 5-fold decrease in emission lifetimes as compared with Cy5-DNA free molecules in the absence of metal. The decrease of lifetime for the Cy5-DNA-Ag particle allowed us to resolve the correlation functions of the two species based on the intensity decays. The increased brightness of the Cy5-DNA-Ag particle as compared to free Cy5-DNA resulted in an increased contribution of Cy5-DNA-Ag to the correlation function of the mixture. These results show that the effects of metal particles on fluorophores can be used to detect the small fractional populations of the metal-bound species in the presence of a larger number of less bright species. Our results also suggest that these bright fluorophores conjugated to silver particles could be used as the fluorescent probes for clinical detection in the biological samples with the high background.