Interactions Between Nanoparticles and Biological Charged Lines: Biological Mimics of Protein-DNA Complexes and Microtubules as Drug Targets

[摘要] DNA-binding proteins use a combination of the following mechanisms to find their DNA target sites: ;;hopping” or ;;jumping” along DNA (3D diffusion), intersegment transfer, sliding (1D diffusion), and site-specific recognition. In particular, the process of sliding is not well understood. It has been hypothesized that while sliding, proteins are ;;loosely” associated with DNA via electrostatic interactions between cationic residues on the protein and anionic phosphate groups on the DNA backbone. To testthis hypothesis, a biomimetic model of sliding was created in which the protein was replaced with cationic particles and the DNA with anionic ;;linear” molecules.The model system utilized in this dissertation was a nanoparticle-microtubule system. Microtubules were chosen because like DNA, they are ;;linear”, negatively-charged biopolymers. Using total internal reflection fluorescence microscopy (TIRFM), it was found that aggregated cationic particles can slide along microtubules.Accordingly, it was hypothesized that the roughened surface of the aggregates mimics the protein conformation complementarity occurring in the cell, and that this complementarity and the juxtaposition of cationic residues within the protein’s DNA binding pocket are crucial to protein sliding.Next, specific binding site recognition was incorporated into the model based on paclitaxel.Paclitaxel is known to bind microtubules and hyperstabilize them.For this cytotoxic property,it is marketed as an anti-cancer drug,although it causes detrimental side effects due to its water insolubility and promiscuity.Accordingly,testing the microtubule binding properties of the paclitaxel-conjugated nanoparticles was of interest.Using TIRFM and transmission electron microscopy (TEM), it was found that paclitaxel-conjugated G5 PAMAM dendrimers affect microtubules by: (1) promoting polymerization; (2) stabilizing microtubules; and (3) bundling microtubules. The latter is independent of paclitaxel and due instead to a combination of electrostatic interactions involving protonatable amines in the dendrimer core, and hydrophobic interactions between the fluorescent labels on the dendrimer (Cy5) and tubulin (TMR). These results warrant further investigation into the toxicity of thecationic dendrimer core before further consideration as paclitaxel delivery platforms.Finally, it is demonstrated that paclitaxel-conjugated gold nanoparticles also showpromise as targeted delivery platforms as they polymerize, stabilize, and bundle microtubules in a paclitaxel-dependent manner.

[发布机构] University of Michigan

[效力级别] Targeted Drug Delivery

[关键词] Nanotechnology;Targeted Drug Delivery;Nanoparticle Toxicity;Microtubules;Paclitaxel;Biosensing and Bioactuation;Molecular;Cellular and Developmental Biology;Science;Cellular & Molecular Biology