Nanostructured supramolecular arrays based on dendrimers using DNA: Design, synthesis and biological evaluation.
Choi, Youngseon
2005
Abstract
This dissertation describes a series of experiments that synthesize and characterize a new type of supramolecular array. These molecules are based on dendrimers linked together with complementary DNA oligonucleotides. The arrays have a good range of applications including cancer cell specific drug targeting. Three specific studies were designed to test the hypothesis that complementary DNA oligonucleotides would self-assemble different single-functional dendrimers to form functional supramolecular clusters. First, in order to produce a library of functionalized dendrimers, generation 5 polyamidoamine (PAMAM) dendrimers were first partially acetylated, then conjugated with either imaging agents (fluorochromes; FITC, 6-TAMRA, and AF488) or targeting molecules (folic acid). The molecular characteristics of each functionalized dendrimers were characterized by UV-Vis, NMR, GPC, HPLC, and MALDI-TOF MS to ascertain their molar extinction, degree of functionalization, molecular weight and molecular weight distribution. Secondly, a synthetic method to covalently attach DNA oligonucleotides to the partially acetylated dendrimers was developed based on phosphoramidate chemistry. This chemistry was then applied to attach three sets of complementary oligonucleotides of different lengths (34, 50, 66 bases-long) to partially acetylated generation 7 and 5 PAMAM dendrimers. The size and the shape of the DNA-linked nanostructure were characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). Finally, we extended our strategy of DNA-based assembly to the construction of dimers of different single-functionalized dendrimers designed for cancer cell specific targeting. Three different batches of DNA-linked dendrimers consisting of imaging dendrimers (G5-FITC, G5-6-TAMRA, or G5-AF488) coupled to targeting dendrimer (G5-FA) consistently targeted KB cells overexpressing the high affinity folate receptor with maximum binding at 100 nM and an apparent binding affinity of ∼ 50 nM and internalized into the cells after 1 hour incubation at 37°C <italic>in vitro</italic>. Preliminary <italic>in vivo </italic> experiments using SCID mice showed that the clusters accumulated in tumor cells expressing folic acid receptors 16 and 48 hours after injection. These results confirmed our hypothesis and demonstrated our ability to design and produce supramolecular arrays of dendrimers using DNA duplex formation. This DNA-linked dendrimer supramolecular array provides a potential platform for developing combinatorial targeted therapeutics in the future.Subjects
Arrays Based Biological Dendrimers Design Dna Evaluation Nanostructured Polyamidoamine Supramolecular Synthesis Using
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