Understanding DNA condensation by low generation (G0/G1) and zwitterionic G4 PAMAM dendrimers.

摘要

Cationic polymers have shown potential as gene delivery vectors due to their ability to condense DNA and protect it from cellular and restriction nucleases. Dendrimers are hyperbranched macromolecules with precisely defined molecular weights and highly symmetric branches stemming from a central core. The nanosize, tunable surface chemistries and ease of surface functionalization has made dendrimers an attractive alternative to conventional linear polymers for DNA delivery applications. The commercially available, cationic dendrimer poly(amidoamine) or PAMAM is the most widely studied dendrimer for use as a gene delivery vector. The aim of this dissertation is to provide an increased understanding of the packaging and forces within PAMAM--DNA complexes.;In Chapter 4, we will discuss the effect of molecular chain architecture on DNA-DNA intermolecular forces by examining DNA condensed by low generation (G0 & G1) PAMAM and comparing them to comparably charged linear arginine peptides. Using osmotic stress coupled with X-ray scattering, we are able to determine the structure and forces within dendrimer-DNA complexes, or dendriplexes. We show that PAMAM--DNA assemblies display significantly different physical behavior than linear cation--DNA assemblies. In Chapter 5, we examine the role of pH on condensation in these same low generation PAMAM-DNA complexes. PAMAM dendrimers have both terminal primary amines and internal tertiary amines with different pKas of approximately 9 and 6, respectively. We show changes in the pH at condensation greatly influence the resulting packaging as well as the resulting phase behavior for PAMAM dendriplexes. In Chapter 6, we examine the packaging of DNA by G4 PAMAM as a function of the percent zwitterionic modification. Many cationic polymers, including PAMAM, have shown high transfection efficiency in cell culture and potential for in vitro and in vivo applications, but its development is hindered by cytotoxicity in many cell lines and tissues. We hypothesize that zwitterionic PAMAM (zPAMAM) represent a new means to tune polymer-DNA interactions through PAMAM surface charge potentially enhancing intracellular unpackaging while reducing cellular toxicity. These zPAMAM complexes are currently under investigation for their potential as safer and more efficient materials for DNA delivery.