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. Moreover, PAMAM dendrimers exhibit greater biocompatibility than other dendrimer families, perhaps due to the combination of surface amines and interior amide bonds; these bonding motifs are highly reminiscent of innate biological chemistry and endow PAMAM dendrimers with properties similar to that of globular proteins. The relative ease/low cost of synthesis of PAMAM dendrimers (especially relative to similarly-sized biological molecules such as proteins and antibodies), along with their biocompatibility, structural control, and functionalizability, have made PAMAMs viable candidates for application in drug development, biochemistry, and nanotechnology.
Synthesis
Divergent synthesis
Divergent synthesis refers to the sequential "growth" of a dendrimer layer by layer, starting with a core "initiator" molecule which contains functional groups capable of acting as active sites in the initial reaction. Each subsequent reaction in the series increases the number of available surface groups exponentially. Core molecules which give rise to PAMAM dendrimers can vary, but the most basic initiators are ammonia and ethylene diamine. Outward growth of PAMAM dendrimers is accomplished by alternating between two reactions: Michael addition of the amino-terminated surface onto methyl acrylate, resulting in an ester-terminated outer layer, and Coupling with ethylene diamine to achieve a new amino-terminated surface. Each round of reactions forms a new "generation", and PAMAM dendrimers are often classified by generation number; the common shorthand for this classification is "GX" or "GX PAMAM", where X is a number referring to the generation number. The first full cycle of Michael addition followed by coupling with ethylene diamine forms Generation 0 PAMAM, with subsequent Michael additions giving rise to "half" generations, and subsequent amide coupling giving rise to "full" (integer) generations.
With divergent synthesis of dendrimers, it is extremely important to allow each reaction to proceed to completion; any defects caused by incomplete reaction or intramolecular coupling of new surface amines with unreacted methyl ester surface groups could cause "trailing" generations, stunting further growth for certain branches. 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. As generation number increases, it becomes more difficult to produce pure products in a timely fashion due to steric constraints. As a result, synthesis of higher-generation PAMAM dendrimers can take months.
Convergent synthesis Convergent synthesis of a dendrimer begins with what will eventually become the surface of the dendrimer and proceeds inward. The convergent synthetic approach makes use of orthogonal protecting groups (two protecting groups whose deprotection conditions will not remove one another); this is an additional consideration not present when using a divergent approach. The figure below depicts a general scheme for a convergent synthetic approach.
Convergent synthesis as shown above begins with the dendritic subunit composed of reactive "focal group" A and branched group B (B can be multiply branched in the most generalized scenario, but PAMAMs only split once at each branching point). First, A is orthogonally protected and set aside for further reactions. B is also orthogonally protected, leaving the unprotected A on this molecule to couple with each of the unprotected B groups from the initial compound. This results in a new higher-generation species that is protected on both A and B. Selective deprotection of A yields a new molecule which can again be coupled onto the original monomer, thus forming another new generation. This process can then be repeated to form more and more layers.
Note that the black protecting groups for group B represent what will become the outermost layer of the final molecule, and remain attached throughout the synthetic process; their purpose is to guarantee that propagation of dendrimer growth can take place in a controlled fashion by preventing unwanted side reactions. In forming each new layer, the number of AB couplings is restricted to two, in sharp contrast to the divergent synthetic approach, which involves exponentially more couplings per layer. Incomplete reaction products (single addition adduct, unreacted starting materials) will have a markedly different molecular weight from the desired product, especially for higher-generation compounds, making purification more straightforward. The reactive focal group A must be terminated onto a final acceptor at some point during the synthetic process; until then, each compound can only be considered a dendron and not a full dendrimer (see page for disambiguation). An advantage to synthesizing dendrons with focal group A as a chemical handle is the ability to attach multiple equivalents of the dendron to a polyfunctional core molecule; changing the core element does not require rebuilding the entire dendrimer. In the case of PAMAM, the focal points of convergently synthesized fragments have been used to create unsymmetrical dendrimers as well as dendrimers with various core functionalization. Since each successive generation of dendron becomes bulkier, with final attachment to the dendrimer core being the most prohibitive step of all, steric constraints can severely impact yield.
Toxicity
in vitro
It has been established that cationic macromolecules in general destabilize the cell membrane, which can lead to lysis and cell death. The common conclusion present in current work echoes this observation: increasing dendrimer molecular weight and surface charge (both being generation-dependent) increases their cytotoxic behavior.
Initial studies on PAMAM toxicity showed that PAMAM was less toxic (in some cases, much less so) than related dendrimers, exhibiting minimal cytotoxicity across multiple toxicity screens, including tests of metabolic activity (MTT assay), cell breakdown (LDH assay), and nucleus morphology (DAPI staining). However, in other cell lines, the MTT assay and several other assays revealed some cytotoxicity. These disparate observations could be due to differences in sensitivity of the various cell lines used in each study to PAMAM; although cytotoxicity for PAMAM varies among cell lines, they remain less toxic than other dendrimer families overall.
More recently, a series of studies by Mukherjee et al. have shed some light on the mechanism of PAMAM cytotoxicity, providing evidence that the dendrimers break free of their encapsulating membrane (endosome) after being absorbed by the cell, causing harm to the cell's mitochondria and eventually leading to cell death. Further elucidation of the mechanism of PAMAM cytotoxicity would help resolve the dispute as to precisely how toxic the dendrimers are.
In relation to neuronal toxicity, fourth generation PAMAM has been shown to break down calcium transients, altering neurotransmitter vesicle dynamics and synaptic transmission. All of the above can be prevented by replacing the surface amines with folate or polyethylene glycol.
It has also been shown that PAMAM dendrimers cause rupturing of red blood cells, or hemolysis. Thus, if PAMAM dendrimers are to be considered in biological applications that involve dendrimers or dendrimer complexes traveling through the bloodstream, the concentration and generation number of unmodified PAMAM in the bloodstream should be taken into account.
in vivo
To date, few in-depth studies on the in vivo behavior of PAMAM dendrimers have been carried out. 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. Nonetheless, the fate and transport of unmodified PAMAM dendrimers is an important case study as any biological applications could involve unmodified PAMAM as a metabolic byproduct. In the only major systematic study of in vivo PAMAM behavior, injections of high levels of bare PAMAMs over extended periods of time in mice showed no evidence of toxicity up through G5 PAMAM, and for G3-G7 PAMAM, low immunogenicity was observed. These systemic-level observations seem to align with the observation that PAMAM dendrimers are not extremely cytotoxic overall; however, more in-depth studies of the pharmacokinetics and biodistribution of PAMAM are required before a move toward in vivo applications can be made.
Surface modification One unique property of dendrimers such as PAMAM is the high density of surface functional groups, which allow many alterations to be made to the surface of each dendrimer molecule. In putative PAMAM dendrimers, the surface is rife with primary amines, with higher generations expressing exponentially greater densities of amino groups. Although the potential to attach many things to each dendrimer is one of their greatest advantages, the presence of highly localized positive charges can be toxic to cells. Surface modification via attachment of acetyl and lauroyl groups help mask these positive charges, attenuating cytotoxicity and increasing permeability to cells. Thus, these types of modifications are especially beneficial for biological applications. Secondary and tertiary amino surface groups are also found to be less toxic than primary amino surface groups, suggesting it is charge shielding which has major bearing on cytotoxicity and not some secondary effect from a particular functional group. Furthermore, other studies point to a delicate balance in charge which must be achieved to obtain minimal cytotoxicity. 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 dendrimers with nonpolar internal components have also been shown to induce hemolysis.
Applications Applications involving dendrimers in general take advantage of either stuffing cargo into the interior of the dendrimer (sometimes referred to as the "dendritic box"), or attaching cargo onto the dendrimer surface. PAMAM dendrimer applications have generally focused on surface modification, taking advantage of both electrostatic and covalent methods for binding cargo. Currently, major areas of study using PAMAM dendrimers and their functionalized derivatives involve drug delivery and gene delivery.
Drug delivery