page_title: Poly(amidoamine)
section_title: Gene therapy
hierarchical_section_title: Poly(amidoamine) / Applications / Gene therapy
caption_attribution_description: Rough scheme of the mechanism of PAMAM-DNA interaction.
context_page_description: Poly(amidoamine), or PAMAM, is a class of dendrimer which is made of repetitively branched subunits of amide and amine functionality. PAMAM dendrimers, sometimes referred to by the trade name Starburst, have been extensively studied since their synthesis in 1985, and represent the most well-characterized dendrimer family as well as the first to be commercialized. Like other dendrimers, PAMAMs have a sphere-like shape overall, and are typified by an internal molecular architecture consisting of tree-like branching, with each outward 'layer', or generation, containing exponentially more branching points. This branched architecture distinguishes PAMAMs and other dendrimers from traditional polymers, as it allows for low polydispersity and a high level of structural control during synthesis, and gives rise to a large number of surface sites relative to the total molecular volume.
context_section_description: The discovery that mediating positive charge on PAMAM dendrimer surfaces decreases their cytotoxicity has interesting implications for DNA transfection applications. Because the cell membrane has a negatively charged exterior, and the DNA phosphate backbone is also negatively charged, the transfection of free DNA is not very efficient simply due to charge repulsion. However, it would be reasonable to expect charged interactions between the anionic phosphate backbone of DNA and the amino-terminated surface groups of PAMAM dendrimers, which are positively ionized under physiological conditions. This could result in a PAMAM-DNA complex, which would make DNA transfection more efficient due to neutralization of the charges on both elements, while the cytotoxicity of the PAMAM dendrimer would also be reduced. Indeed, several reports have confirmed PAMAM dendrimers as effective DNA transfection agents.
  When the charge balance between DNA phosphates and PAMAM surface amines is slightly positive, the maximum transfection efficiency is obtained; this finding supports the idea that the complex binds to the cell surface via charge interactions. A striking observation is that "activation" of PAMAM by partial degradation via hydrolysis improves transfection efficiency by 2-3 orders of magnitude, providing further evidence supporting the existence of an electrostatically coupled complex. The fragmentation of some branches of the dendrimer is thought to loosen up the overall structure (fewer amide bonds and space constraints), which would theoretically result in better contact between the dendrimer and DNA substrate because the dendrimer is not forced into a rigid spherical conformation due to sterics. This in turn results in more compact DNA complexes which are more easily endocytosed. After endocytosis, the complexes are subjected to the acidic conditions of the cellular endosome. The PAMAM dendrimers act as a buffer in this environment, soaking up the excess protons with multitudes of amine residues, leading to the inhibition of pH-dependent endosomal nuclease activity and thus protecting the cargo DNA. The tertiary amines on the interior of the dendrimer can also participate in the buffering activity, causing the molecule to puff up; additionally, as the PAMAMs take on more and more positive charge, fewer of them are required for the optimal PAMAM-DNA interaction, and free dendrimers are released from the complex. Dendrimer release and swelling can eventually lyse the endosome, resulting in release of the cargo DNA. The activated PAMAM dendrimers have less spatial barrier to interior amine protonation, which is thought to be a major source of their advantage over non-activated PAMAM.
  In the context of existing approaches to gene transfer, PAMAM dendrimers hold a strong position relative to major classical technologies such as electroporation, microinjection, and viral methods. Electroporation, which involves pulsing electricity through cells to create holes in the membrane through which DNA can enter, has obvious cytotoxic effects and is not appropriate for in vivo applications. On the other hand, microinjection, the use of fine needles to physically inject genetic material into the cell nucleus, offers more control but is a high-skill, meticulous task in which a relatively low number of cells can be transfected. Although viral vectors can offer highly specific, high-efficiency transfection, the generation of such viruses is costly and time-consuming; furthermore, the inherent viral nature of the gene transfer often triggers an immune response, thus limiting in vivo applications. In fact, many modern transfection technologies are based on artificially assembled liposomes (both liposomes and PAMAMs are positively charged macromolecules). Since PAMAM dendrimers and their complexes with DNA exhibit low cytotoxicity, higher transfection efficiencies than liposome-based methods, and are effective across a broad range of cell lines, they have taken an important place in modern gene therapy methodologies. The biotechnology company Qiagen c
caption: Surface amine residues on PAMAM dendrimers bind to the phosphate backbone of nucleic acids through charged interactions (right, inset). Typically, G6-7 PAMAM dendrimers are used for gene transfection; these dendrimers are typically 6-10nm in length (spanning ~20-30 base pairs) and have a molecular mass of 30-50kDa.[25]
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