NSTI BioNano 2010

Cellular Binding and Uptake of Aptamer-Targeted Delivery Systems

W.T. Al- Jamal, J. Van den Bossche, B. Tian, A. Nunes, C. Fabbro, M. Prato, A. Biancoc, K. Kostarelosa
The School of pharmacy, UK

Keywords: CNT, liposomes, aptamers, targeting


Aptamers are DNA or RNA sequences, that can be selected (mainly via the SELEX methodology) to recognize a wide variety of targets such as proteins, lipids, nucleic acids and sugars with high affinity and selectivity extracellularly as intracellularly. In contrast to antibodies, aptamers have shown higher stability upon storage and lower immunogenicity in vivo. MUC-1 antigens are known markers that are overexpressed and aberrantly glycosylated on the membrane of various cancer cells. To explore the targeting capability of MUC-1 aptamers in cancer therapy, ssDNA 25 bp aptamers were chemically conjugated to liposome and carbon nanotube (CNT) surfaces. Aptamer-targeted liposomes were prepared by conjugating a thiolated aptamer to maleimide-PEG-DSPE2000 micelles and subsequently post-inserted into preformed liposomes. On the other hand, carboxylated multiwalled nanotubes (MWNTs) were conjugated using one-step ligation to amine-functionalised aptamers using 1-[3 -(dimethylamino)propyl]-3-ethylcarbodiimide/N-hydroxysuccinimide (EDCI/NHS). The aptamer chemical conjugation onto the two types of delivery systems was confirmed using polyacrylamide gel. Aptamer targeted liposomes and CNT constructs were further characterized using transmission electron microscopy (TEM), atomic force microscopy (AFM) and thermogravimetric analysis. The specific binding of the aptamer-conjugated delivery systems to MUC-1 receptors in vitro was studied using confocal laser scanning microscopy (CLSM). Fluorescently-labeled aptamer alone exhibited affinity for the plasma membrane of MUC-1 positive cells only. The cellular uptake studies of aptamer- conjugated liposomes and CNT showed interesting and dramatically different results among the two types of delivery systems. Aptamer-conjugated liposomes can specifically target and be uptaken by human breast cancer cells (MCF-7) which overexpress MUC-1 antigens in contrast to human pulmonary adenocarcinoma cells (Calu-6) that have low levels of MUC-1 expression. On the other hand, the MWNT-aptamer conjugates were delivered into the cytosol of all cell types in a MUC-1 independent manner. In conclusion, we clearly demonstrated that the binding affinity of MUC-1 aptamers is critically determined by the properties of the delivery systems. Aptamer conjugation to liposomes can offer a new class of targeted system to deliver intracellularly a wide range of therapeutics for in vitro and in vivo applications. On the other hand, carbon nanotubes can be an excellent tool for the efficient intracellular delivery of therapeutic aptamers that may exhibit affinity for various intracellular targets
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