Poly(amidoamine)

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.

These impurities are difficult to remove when using the divergent synthetic approach because the molecular weight, physical size, and chemical properties of the defective dendrimers are very similar in nature to the desired product.

In relation to neuronal toxicity, fourth generation PAMAM has been shown to break down calcium transients, altering neurotransmitter vesicle dynamics and synaptic transmission.

This could be in part due to the diverse behavior of PAMAMs depending on surface modification (see below), which make characterization of their in vivo properties largely case-dependent.

In putative PAMAM dendrimers, the surface is rife with primary amines, with higher generations expressing exponentially greater densities of amino groups.

Surface modification via attachment of acetyl[18] and lauroyl[19] groups help mask these positive charges, attenuating cytotoxicity and increasing permeability to cells.

Hydrophobic interactions can also cause cell lysis, and PAMAM dendrimers whose surfaces are saturated with nonpolar modifications such as lipids or polyethylene glycol (PEG) suffer from higher cytotoxicity than their partially substituted analogues.

PAMAM dendrimer applications have generally focused on surface modification, taking advantage of both electrostatic and covalent methods for binding cargo.

The discovery that mediating positive charge on PAMAM dendrimer surfaces decreases their cytotoxicity has interesting implications for DNA transfection applications.

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.

A striking observation is that "activation" of PAMAM by partial degradation via hydrolysis improves transfection efficiency by 2-3 orders of magnitude,[23] providing further evidence supporting the existence of an electrostatically coupled complex.

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.

[25] 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.

[25] 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,[16] they have taken an important place in modern gene therapy methodologies.

The biotechnology company Qiagen currently offers two DNA transfection product lines (SuperFect and PolyFect) based on activated PAMAM dendrimer technology.

Although the dendrimers have proved to be highly efficient and non-toxic in vitro, the stability, behavior, and transport of the transfection complex in biological systems has yet to be characterized and optimized.

A general scheme for the divergent synthesis of PAMAM dendrimers, with ethylene diamine as a core initiator. The scheme is color-coded by generation number, with the red ethylene diamine core serving as initiator core, orange as Generation 0 and orange/green as Generations 1, respectively. The scheme shown is currently the most widely adopted approach in commercial syntheses of PAMAM. [ 5 ]
A generalized scheme outlining the use of orthogonal protecting groups for convergent synthesis of PAMAM dendrimers.
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 ]
PAMAM dendrimers can be "activated" for gene transfer applications via hydrolysis accelerated by heat, a process which can be thought of as similar to shearing bushes. During this process, amide bonds are broken and replaced with carboxyl groups (see inset), causing some branches of the dendrimer to fall off. The overall molecular mass of the dendrimer is reduced by 20-25%, and the result is a more flexible dendrimer with transfection efficiencies improved by 2-3 orders of magnitude. [ 25 ]