Agents able to bind tightly and selectively to disease markers can greatly benefit disease diagnosis and therapy. Aptamers are functional molecules, usually DNA or RNA oligonucleotides, with the appropriate sequence and structure to form a complex with a target molecule. MUC1 is a well-known tumour marker present in a variety of malignant tumours and it has been a target of interest for many years. In this work we report the selection of DNA aptamers that bind with high affinity and selectivity to the MUC1 peptides. Combinatorial chemistry techniques based on the SELEX methodology were used for the identification of the specific aptamers. These were selected from an initial library containing a 25-base-long variable region, resulting in 4²⁵ random sequences of single-stranded DNA molecules, for their ability to bind to synthetic forms of MUC1. Ten rounds of in vitro selection were performed enriching for MUC1 binding. By round ten more than 90% of the pool of sequences consisted of MUC1-binding molecules. Selected aptamer families were cloned, sequenced and found to be unique, sharing no sequence consensus. The binding properties of these aptamers were quantitated by enzyme-linked immunosorbent assay and surface plasmon resonance, whereas their specificity for MUC1-expressing cancer cells has been validated using fluorescent microscopy. Aptamers offer significant advantages over existing antibody-based recognition procedures in that they offer higher binding affinity (higher retention/reduced dissociation) and specificity to the target (ability to determine variations on the protein target down to single amino acid changes), higher selectivity against mutated protein epitopes and potentially reduced immunogenicity and increased tumour penetration associated with their size.

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells.

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Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer-antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC50 value of 25 microg/ml, a detection limit of 1 microg/ml and a linear range between 8 and 100 microg/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours.

Lastly, the benefits of dietary omega-3 fatty acids and their mechanisms of action leading to reduced cancer risk and impeded cancer cell growth are mentioned. Finally, a proposal is put forward, suggesting a novel and integrated approach in viewing the molecular mechanisms and complex interactions responsible for the involvement of AA metabolites in carcinogenesis and the protective effects of omega-3 fatty acids in inflammation and tumour prevention.

Targeted radiopharmaceuticals offer the possibility of improved tumor imaging and radiotherapy, with reduced side effects. A variety of monoclonal antibodies and antibody fragments have previously been successfully radiolabeled and used in diagnostic imaging and targeted radiotherapy of cancer. Many such antibodies have been shown to recognize the well-characterized MUC1 tumor marker and have recently been in clinical trials. Furthermore, a number of chelators have been synthesized and are currently used as radiopharmaceuticals for imaging and therapy. We now report the synthesis of a novel, cyclen-based ligand with a sulfur-containing arm that offers increased stability of the ligand-metal complex. We have coupled this ligand with previously selected aptamers to the MUC1 tumor marker to generate a novel targeted radiopharmaceutical with improved properties. We have tested the complex against known, commercially available chelators such as MAG3 in model breast cancer systems. To improve the pharmacokinetic properties of the aptamer-based targeted radiopharmaceutical, we have generated multi-aptamer complexes around a central chelator. Such multi-aptamer complexes have increased retention of the complex in circulation, without affecting the lack of immunogenicity of the complex or altering its superior tumor penetration properties.

Tc, for the potential use as radiopharmaceuticals for diagnostic imaging of breast cancer. Methods:The conjugation was achieved in high yield using standard peptide coupling reactions between an amino modification on the aptamer and the activated carboxylic group on the ligands. The retention of the affinity of the MAG2 modified AptA for the MUC1 protein core was confirmed using a FID binding assay. The 2 labelled aptamers were separated from free 99m Tc using microcon filter separation and monitored by HPLC at all stages, to ensure that only radiolabelled aptamers were produced. The biodistribution properties of the two aptamer-radionuclide conjugates were analysed in MCF-7 tumour bearing mice and compared. Tc. The radiolabelled aptamers showed different tumour uptake and clearance, but will require further development prior to diagnostic use. Results

Aptamers are characterized by a rapid renal clearance leading to a short in vivo circulating half-life. In order to use aptamers as anticancer therapeutic agents, their exposure time to the tumor has to be enhanced via increasing residency in the bloodstream. A way to achieve this goal is by conjugating the aptamer to poly(ethylene glycol) (PEG). Herein, we present the conjugation of a bifunctionalized anti-MUC1 aptamer (NH(2)-AptA-SR) with the (99m)Tc coordinating moiety MAG2 and either a conventional branched PEG or the comb-shaped PolyPEG via a two-step synthesis. The isolated products were radiolabeled with (99m)Tc and their biodistribution and tumor-targeting properties in MCF-7 tumor bearing mice were analyzed and compared.