Pharma Tips

Dendrimers -Novel Drug Delivery System

By: Pharma Tips | Views: 8919 | Date: 18-Jun-2010

Dendrimer is a highly branched polymer, as shown in the schematic below, and consists of a core where a monomer unit is attached.


I. Linear 
II. Crosslinked 
III. Branched 


Dendrimer is a highly branched polymer, as shown in the schematic below, and consists of a core where a monomer unit is attached. 

Dendrimers are large and complex molecules with very well-defined chemical structures. From a polymer chemistry point of view, dendrimers are nearly perfect monodisperse (basically meaning of a consistent size and form) macromolecules with a regular and highly branched 3 dimensional architecture. 
They consist of three major architectural components: 
1. Core
2. Branching Unit  
3. End groups

Dendrimers are produced in an iterative sequence of reaction steps, in which each additional iteration leads to a higher generation dendrimer. The creation of  dendrimers, using specifically-designed chemical reactions, is one of the best examples of controlled hierarchical synthesis, an approach that allows the 'bottom-up' creation of complex systems. 
Each new layer creates a new 'generation', with double the number of active sites (called end groups) and approximately double the molecular weight of the previous generation. One of the most appealing aspects of technologies based on dendrimers is that it is relatively easy to control their size, composition and chemical reactivity very precisely.
It has been suggested that the need to repeat reaction steps many times could lead to high manufacturing costs and thus hinder commercialization. However, the synthesis is, in most cases, just the iterative sequence of two straightforward chemical reactions, the reactants are normally cheap chemicals, and with each step the product practically doubles its weight. Consequently, the manufacture of dendrimers can be a cost effective process and large-scale production is, in general, technically and economically viable. PPI and PAMAM dendrimers (explained later) are already being produced on a large scale by companies such as DSM and Dendritech, and can be ordered through the Sigma-Aldrich catalog, like any other common chemical compound.

Derivatives of the classical well-defined dendrimers are currently being evaluated as potential alternatives offering lower production costs. Dow Corning Corporation, in collaboration with the Michigan Molecular Institute, has developed a new class of dendrimers, which contain PAMAM (see later) interiors and organosilicon (OS) exteriors. 

Dendrimer Structure, Courtesy of Dendritech.

These nanobuilding blocks can form films, membranes and coatings with controlled hydrophilic (water-loving) and hydrophobic (water-hating) domains. They can be applied to the preparation of printed wire boards, sensors, catalysts, and drug delivery systems.

Fig.,Dendritic box. Courtesy of the Eindhoven University of Technology, the Netherlands 

Hyperbranched structures are built in a similar way to dendrimers and they have a similar structure, but their architecture is not as precisely controlled (not all the molecules in a batch look the same: they miss branches; there are more holes in the structure and fewer active chemical groups at the surface of the molecule). To make these 'imperfect' molecules, a far less rigorous optimization of the production process is needed (less care is required in ensuring that the synthesis steps have been completed). In some cases far fewer   steps are necessary to produce hyperbranched structures, which leads to reduced production costs.
For many applications dendrimers do not have to be perfect to be useful and it is important to remember that hyperbranched structures could replace dendrimers in many such cases. Think, for example, of additives for lowering viscosity (where the requirement is simply for globular structures) or decontamination agents (where larger holes could even be an advantage for binding larger molecules). On the contrary, if  multivalency (a large number of active chemical groups located at the surface of the dendrimer) and well-defined structure are needed, as is the case for biomedical applications, dendrimers are much better candidates.

The cavities present in dendrimers can be used as binding sites for small guest molecules, analogous to the molecules, analogous to the catalysts, or promoters of chemical reactions) work in living organisms.

The potential of dendrimers as hosts (containers) for small molecules was strikingly demonstrated in 1994 by Bert Meijer, chemistry professor at Eindhoven University of Technology in the Netherlands. The 'dendritic box' was constructed by building shell around the soft-core dendrimer once a small 'probe' molecule was encapsulated into the dendrimer. This structure yielded a molecular container of nanoscopic dimensions.
Dendrimers are generally roughly spherical molecules with very well defined size and shape. This physical characteristic, combined with their thermally decomposable organic nature, makes them especially suitable for use as porogen (a pore-generating material). This can be applied in the creation of foams with optimized nanoscale pore sizes and distributions, such as synthetic zeolites for catalysis or low dielectric constant materials (in simple terms, good insulators) for use in ever-shrinking integrated circuits. 

They have also been used as templates to obtain larger structures with particular characteristics. Recently, the first example of the formation of amorphous calcium carbonate by artificial methods was reported. The structures created used dendrimers as an organic template in an attempt to imitate nature in the way it constructs shells and bones (an example of the power of this sort of nanostructuring is nacre, or mother-of-pearl, which is extremely tough despite being largely made of the same material as chalk). However, the process is not yet completely understood and applications in the manufacture of artificial bone currently look remote, with other approaches a lot closer to market, such as the coating of artificial joints with nanograins of hydroxyapatite.

Dendrimers can also be grown around a template (acting as the core) and then cross-linked to fix the structure. The core can then be removed to create a cavity that selectively binds to the original template. This creates a structure that is imprinted at the molecular level and that has a specific binding site (like an artificial antibody) that can separate enantiomers (mirror image versions of a molecule, only one of which usually has the desired function), remove pollutants or catalyze reactions.

The fact that dendrimers are generally depicted as spherical molecules can be misleading. Dendrimers generally have a globular shape when they are held in a good solvent, or in a bulk material, or when the end groups are so bulky that they get in the way of each other's ability to move around, making the structure rigid (this is known as steric hindrance). 

However, 'bare' dendrimers are very flexible structures. When secondary interactions are introduced into the system (for example, those based on the affinity of the end groups for a particular surface or hydrophobic (water-hating)interactions that can lead to self-assembly), dendrimers have the ability to completely change their confor mation to form layers or even self-assembled lipid-like structures. They can transform their shape from spherical to almost completely flat, if the interactions with the surface are adequate.

Self-organization, or self-assembly, is an approach to the construction of new nanomaterials, such as coatings, non-linear optic materials, polymer electronics, etc., that is receiving a lot of attention. The paradigm is being explored extensively in areas such as sol–gel chemistry and self-assembled monolayers. 
Many such applications are geared toward creating bulk materials, coatings being a predominant example. The self-assembly paradigm, however, also offers the possibility of creating discrete functional nanostructures, or even nanodevices, rather than bulk materials.The creation of nanocapsules or quantum dots in liquid phase (as opposed to creating them on a surface), for example, holds great promise for applications such as drug delivery or bioanalysis. Central to many of these approaches is the use of amphiphilic substances, which have hydrophobic (water-hating) and hydrophilic (water-loving) regions that cause them to spontaneously form nanostructures such as capsules, by virtue of one part of the molecule being repelled by the surrounding medium and another part being attracted by it. 

Dendrimers can be amphiphilic (see glossary) and thus can be persuaded to self-organize into nanoscale structures but have the added advantage that they can be designed with a very wide variety of chemical and physical properties. These can be leveraged to produce self-assembled complex multifunctional units that really start to warrant the description of a nanodevice. Some examples will be given later.
Some of the most interesting applications for dendrimers, in a technical and commercial sense, are in the pharmaceutical and biomedical area. Dendrimers can be created that are biologically active or inert, and one of the attractions is that they can be designed for such properties from the bottom up. The molecules are small enough to pass into cells and can be used to deliver substances such as drugs, genetic material or chemical markers right into the cell. James Baker of the University of Michigan in Ann Arbor has built multi-purpose dendrimers that can deliver a drug, document that the drug is there and report back on the cell's response. This is a real example of a multifunctional nanodevice.
On a slightly simpler level, the ability to engineer all sorts of behavior into dendrimers holds promise for a number of biotechnology and medical applications. For example, dendrimers have been deployed as transfection agents (carrying and incorporating foreign genetic material into cells), for mediating transport across cell membranes, or for binding particles to inhibit an immune response.

The first monomer unit, in this example, has a functionality of three with one reactive site attached to the core or focal point and the other two making up a branching unit. This is considered the first generation or G1. The branching unit is then reacted with further monomer to produce G2 and a molecule with four end groups. 
For a given functionality (f ) the number of end groups scales as 
NE = [f-1]g
where g is the generation number; 0 (the core only), 1, 2, … Likewise, the dendrimer molecular mass (M) scales as 
M ~ [f-1]g
An example of a poly(benzyl ether) dendrimer is given below. This is a G4 dendrimer with an -OH core. 
Dendrimers show some unique properties, one is the intrinsic viscosity going through a maximum at the fourth generation (see e.g. T. H. Mourey, S. R.Turner, M. Rubenstein, J. M. J. Fréchet, C. J. Hawker and K. L. Wooley,  “Unqiue Behaviour of Dendritic Macromolecules: Intrinsic Viscosity of polyether Dendrimers,” Macromolecules, 25 (1992) 2401-2406). This is sometimes denoted as a globular transition which may be more apparent below. 
A reference to this type of dendrimer is  C. J. Hawker and J. M. J. Fréchet,  “Preparation of polymers with controlled molecular architecture.   A new convergent approach to dendritic macromolecules,” J. Am. Chem. Soc., 112 (1990) 7638-7647.
General references to dendrimers are:  J. M. J. Fréchet,  “Functional Polymers and Dendrimers: Reactivity,Molecular Architecture, and Interfacial Energy,” Science, 263 (1994) 1710-1715. 
D. A. Tomalia,  “Dendrimer Molecules,” Scientific American, May (1995) 62-66. B. I. Voit,  “Dendritic polymers:  from aesthetic macromolecules to commercially interesting materials,” Acta Polymer, 46 (1995) 87-99. 

Through Einstein's relation for suspension viscosity the above can be rationalized. Einstein showed the intrinsic viscosity ([]) for hard spheres is given by 
[] = 5/2 /c
where  is the volume fraction of polymer in a solvent (ratio of polymer volume to total volume) and c the polymer concentration (ratio of polymer mass to total volume). Thus, [] is merely the ratio of polymer volume (V) to polymer mass (M). 
The dendrimer volume is taken to a first approximation as 
V ~ g3
or the linear dimension grows steadily with generation number thereby making the volume increase accordingly. Using the above molecular mass scaling one can find 
[] ~ V/M ~ g3/[f-1]g
This equation has a maximum (amazingly) at G4 which is where Mourey et al. found their maximum for poly(benzyl ether) dendrimers. In general, the intrinsic viscosity maximum (gmax) is given by 
gmax = 3/ln(f-1)
for any functionality. 
This is a crude calculation, yet, does serve to show the reason why the peculiar intrinsic viscosity maximum is seen. The molecular volume can at most scale with g3 while the mass is set by the functionality and scales with a power law in generation number. Eventually the power law become the dominant factor and the intrinsic viscosity decreases, in other words, the volume does not suddenly "collapse," rather the mass increases faster than the volume. A more detailed analysis is given in an appendix below, yet, it does not reveal new insight. 

Note the molecular density ( ~ 1/[]) will asymptotically scale as 
 ~ ln(f-1) [f-1]g
and will thus tend to infinity. This has led to the speculation that dendrimers will not be made above a certain generation as the crowding will become too great. This seems to be the case since dendrimers of generation greater than 7-10 are not reported. (n.b. The range of maximum generation number is due to the various branching units used. Some are more flexible than others and can be made to a larger maximum generation. The work of Mansfield and Klushin (Macromolecules, 26 (1993) 4262-4268) is interesting since they have shown the end groups saturate the entire molecule and are not localized at the molecular periphery. This prediction certainly depends on the branching unit flexibility, yet, is provocative. Further work by Mansfield shows at high generation that the molecular density is approximately constant (Macromolecules, 33 (2000) 8043-8049).) Appendix - Generation number for a maximum in the intrinsic viscosity. The volume is written as 
V ~ g+1
where  is an exponent assumed equal to 2 in the above simple analysis. For linear polymers, this exponent is 1 (Rouse model), 1/2 (Zimm model) and 4/5 (Good solvent) as demonstrated by Doi and Edwards (M. Doi and S.F. Edwards, The Theory of Polymer Dynamics, Oxford (1989)). Of course, a rigid rod polymer will have  equal to 2 also. A dendrimer's molecular mass can be more accurately written as scaling 
M ~ {[f-1]g - 1}/[f-2] + r [f-1]g
where r is the ratio of the terminal to branching unit's molecular mass (0.752 for poly(benzyl ether) dendrimers). This equation has the advantage of reducing to the proper limit when f tends to 2 for linear polymers, M ~ g and is easily proven when l-Hôpital's rule is used. 
The intrinsic viscosity is merely V/M (plus unknown scaling factors) and can be shown to have a maximum at the following generation number 
0 = gmax - {[+1]/ln(f-1)}  {1-1/[{1+r[f-2]}[f-1]gmax]}
Assuming  equal to 2, gmax is found to be 4.19 while the above approximate treatment yielded 4.33. Taking  as a measure of monomer flexibility one finds gmax equal to 2.62 for  equal to 1. 
The intrinsic viscosity maximum appears to be between generation 4 and 5 suggesting poly(benzyl ether) dendrimers are fairly stiff and each generation merely adds onto the molecular dimension in a linear manner. 
Continuing this line of thought could lead to the conclusion that intrinsic viscosity maxima at generation numbers greater than G5 represents imperfect dendrimers as demonstrated now. Fractional powers for f may be used to represent imperfect dendrimers based on the expected chemical reaction. In fact, I will take fractional powers as a model for hyperbranched polymers. Assuming 
f = 2.5, r = 1
one finds the following results   
 gmax
0.5 2.85
0.8 3.72
1.0 4.31
2.0 7.10
So, even if the intrinsic viscosity maximum is at generation 4 it is not entirely clear if a perfect dendrimer is present. Other conclusions can be made based on this model such as a hyperbranched polymer will have a gmax at larger values for a given flexibility compared to a dendrimer. This technique could be used to determine the functionality should a dendrimer and a hyperbranched polymer be made with a given monomer unit. However, we have noted hyperbranched polymers are polydisperse in nature and this may make this technique useless unless the hyperbranched polymer is fractionated.

Each of the three macromolecular architectural classes,  i.e.
(I) Linear, 
(II) Crosslinked, and 
(III) Branched, has spawned rich polymer science. 
These architectural discoveries have been characterized by the emergence ofnew syntheses, structures, phenomena, properties, and products that have dramatically improved the human condition1.In the past five years, nanotechnology initiatives 2 have focused on new synthesis strategies, structures, phenomena, and properties associated with length scales of 1-100 nm. These dimensions encompass biological building blocks (protein, DNA, RNA, etc.) and abiotic application areas (nanophotonics and nanoelectronics) 
This review will focus on an emerging, fourth class of architecture, ‘the dendritic state’, and its convergence with nanotechnology5,6.
Dendritic architecture is one of the most pervasive topologies observed in nature at the macro- and micro-dimensional length scales (i.e. meters to micrometers). At the nanoscale (molecular level), there are relatively few natural examples of this architecture. The most notable are glycogen and amylopectin, macromolecular hyperbranched structures that nature uses for energy storage. In thepolymer field, dendritic topology has now been recognized as a fourth major class of macromolecular architecture7-10. The signature for such a distinction is the unique repertoire of new properties manifested by this class of Polymers  6,8,9,11-15. 
Numerous synthetic strategies have been reported for the preparation of these materials, which have led to a broad range of dendritic structures. Presently, this architectural class consists of four dendritic subclasses: (IVa) random hyperbranched polymers, (IVb) dendrigraft polymers, (IVc) dendrons, and (IVd) dendrimers (Fig. 2). The order of this subset, from IVa to IVd, reflects the relative degree of structural control present in each of these dendritic architectures7,8. The relationship of dendritic polymers to traditional polymer classes can be better understood by reviewing the significant role this new topology plays in the continuum of architectures that reside between the two classical areas of ‘thermoplastic’ and ‘thermoset’ polymers16-18. It is now recognized that a continuum of architectures and properties, Beginning with classical branched polymers, resides between these two areas. In contrast to linear random coil polymers, typical branched structures such as starch or high-pressure poly(ethylenes) are characterized by more than two terminal groups per molecule. Hence, they possess other architecturally driven properties, such as substantially smaller hydrodynamic volumes and different intrinsic viscosities compared to their linear polymer counterparts, yet exhibit unexpected segmental expansion near the ‘theta state’19.

Fig. 1 Dimensional comparison of poly(amidoamine) (PAMAM) dendrimers with an NH3 core (G = 0-7) with biological building blocks (cell, proteins, DNA, lipid bilayer), small molecules, atoms, and the electromagnetic energy spectrum.
Completing this continuum, we may now focus on the intermediary role that class IV dendritic polymers play, both in architecture and properties, as penultimate thermoplastic precursors to class II crosslinked thermoset systems. 
Thermoset polymer pioneers such as Dusek et al.20 have thoroughly examined the critical architectures residing between these traditional thermoplastic and thermoset systems. They conclude that random hyperbranched polymers (IVa) best represent the critical, penultimate thermoplastic architectural precursors that lead to the thermoset state (Fig. 2)21. It is now widely recognized that within the four dendritic subsets, dendrons, dendrimers, and, to a lesser extent, dendrigraft polymers represent higher complexity with extraordinary structure control.
This is in contrast to random hyperbranched polymers, which are statistical distributions of molecular weights and structures much like the first three traditional polymer classes. Therefore, the ‘dendritic state’ may be visualized as a progression from lower order, statistical complexities (i.e. Class I-III) to substantially higher levels of monodisperse, structure-controlled complexity22. 
As such, covalent bridging or crosslinking of these preformed dendron/dendrimer modules would be expected to give rise to a completely new  class (V) of regio-specific bridged (crosslinked in a new way) and more ordered (structure-controlled) complexity. 
Examples of these new architectures have been synthesized and been termed ‘megamers’6,23,75. These new topologies and their unique properties are described later. All dendritic polymers are open, covalent assemblies of branch cells or BCs (Fig. 3). 
They may be organized as very symmetrical, monodisperse arrays as is the case for dendrimers, or as irregular, polydisperse assemblies that typically define random hyperbranched polymers. The respective subclasses and the level of structure control are defined by the propagation methodology used to produce these assemblies, as well as the BC construction parameters determined by the composition of the BC monomers and the nature of the BC excluded volume. 

The excluded volume of the BC is determined by the length of the arms; the symmetry, rigidity, or flexibility; and the branching and rotation angles within each of the BC domains. As shown in Fig. 3, these dendritic arrays of BCs usually manifest covalent connectivity relative to some molecular reference marker or core (I). As such, these BC arrays may be nonideal and polydisperse (e.g. Mw /Mn ≅2-10) as observed for random hyperbranched polymers (IVa), or ideally organized into highly controlled core-shell-type structures as noted for dendrons (IVc) and dendrimers (IVd) (Mw/Mn ≅1.0000-1.05). Dendrigraft polymers reside between these two extremes of structure control, frequently manifesting narrow polydispersities of Mw/Mn ≅1.1-1.5, depending on their mode of preparation (Fig. 3).
Fig. 2 Comparison of polymer architectures, (I) linear, (II) crosslinked, and (III) branched with (IV) dendritic, and their relationship in the transition from the thermoplastic to the thermoset state. The derivation of all architectural classes from classical monomers is noted, whereas dendrons and dendrimers may function as nanoscale monomers in megamers.

Random hyperbranched polymers
Flory first hypothesized dendritic polymer concepts18,24, which are now recogni-zed to apply to statistical or random hyperbranched polymers. However, the first experimental confirmation of dendritic topologies did not produce random hyper-branched polymers but rather the more precise, structure-controlled dendrimer architecture7,8,25,26. 

This work was initiated nearly a decade before the first examples of random hyperbranched polymers were confirmed independently by Gunatillake et al.27 as well as Kim and Webster28,29 in 1988. At that time, Kim and Webster coined the popular term ‘hyperbranched polymers’ that has been widely used to describe this subclass of dendritic macromolecules. 
Hyperbranched polymers are typically prepared by polymerization of ABx monomers. When x is 2 or more, polymerization of such monomers gives highly branched polymers (see Fig. 3), as long as A reacts only with B from another molecule. Reactions between A and B from the same molecule result in termination of polymerization by cyclization. This approach produces hyperbranched polymers with a degree of polymerization n, possessing one unreacted. 
A functional group and [(x-1)n + 1] unreacted B terminal groups. In a similar fashion, copolymerization of A2 and B3, or other such polyvalent monomers, can give hyperbranched polymers30,31 if the polymerization is maintained below the gel point by manipulating monomer stoichiometry or limiting polymer conversion. Random hyperbranched polymers are generally produced by one-pot polymeri-zation of ABx-type monomers or macromonomers involving polycondensation, ring-opening, or polyaddition reactions. Hence, the products usually have broad, statistical molecular weight distributions, as observed for traditional polymers. Over the past decade, literally dozens of new AB2-type monomers have been reported, leading to an enormously diverse array of hyperbranched structures. Some general types include poly(phenylenes) obtained by 
• Suzuki coupling28,29; 
• Poly(phenylacetylenes) prepared by the Heck reaction32; 
• Polycarbosilanes, 
• Polycarbosiloxanes33, and 
• Poly(siloxysilanes) by hydrosilylation34; 
• Poly(ether ketones) by nucleophilic aromatic substitution35; and 
• Polyesters36 or polyethers37 by ring-opening polymerization38.

New advances beyond the traditional AB2 Flory-type monomers have been reported by Fréchet and coworkers39,40. They have introduced the concept of latent AB2 monomers, referred to as self-condensing vinyl polymerizations (SCVP). These monomers, which possess both initiation and propagation properties, may follow two modes of polymerization; namely, polymerization of the double bond (i.e. chain growth) and condensation of the initiating group with the double bond (i.e. step growth). Recent progress involving the derivative process of self-condensing ringopening polymerizations (SCROP) has been reviewed by Sunder et al.41. In addition, the use of enhanced processing techniques, such as pseudo-chain growth by slow monomer addition42, allows somewhat better control of hyperbranched structures41.

Dendrigraft polymers
Dendrigraft polymers are the most recently discovered and currently the least understood subset of dendritic polymers. The first examples were reported in 1991 independently by Tomalia et al.43 and Gauthier and Möller44. Whereas traditional monomers are generally employed in constructing dendrimers, reactive oligomers or polymers are used in protect-deprotect or activation schemes to produce dendrigrafts. 

Consequently, dendrigraft polymers are generally larger structures than dendrimers, grow much faster, and amplify surface groups more dramatically as a function of generational development. Both hydrophilic (e.g. polyoxazolines and poly(ethyleneimines)) and hydrophobic dendrigrafts (e.g. polystyrenes) were reported in these early works. 

The first methodologies involved the iterative grafting of oligomeric reagents derived from living polymerization processes in various iterative, ‘graft-on-graft strategies. By analogy to dendrimers, each iterative grafting step is referred to as a generation. An important feature of this approach is that branch densities, as well as the size of the grafted branches, can be varied independently for each generation. 

Furthermore, by initiating these iterative grafting steps from a point-like core or a linear core, it is possible to produce spheroidal and cylindrical dendrigrafts, respectively. Depending on the graft densities and molecular weights of the grafted branches, ultrahigh molecular weight dendrigrafts (e.g. Mw > 104 kDa) can be obtained at very low generation levels (e.g. G = 3). Dramatic molecular weight enhancements vis-à-vis other dendrimer propagation methodologies are possible using dendrigraft techniques45. 
Further elaboration of these dendrigraft principles has allowed the synthesis of a variety of core-shell-type dendrigrafts, in which elemental composition as well as the hydrophobic or hydrophilic character of the core is controlled independently46.

In general, the methodologies above have involved convergent-type grafting principles, where preformed, reactive oligomers are grafted onto successive branched precursors to produce semicontrolled structures. Compared to dendrimers, dendrigraft structures are less controlled since grafting may occur along the entire length of each generational branch, and the exact branching densities are somewhat arbitrary and difficult to control.

More recently, both Gnanou et al.47,48 and Trollsäs and Hedrick49,50 have developed approaches to dendrigrafts that mimic dendrimer topologies by confining the graft sites to the branch termini for each generation. These methods involve so-called ‘graft from’ techniques and allow better control of branching topologies and densities as a function of generation. 

Topologies produced by these methods are reminiscent of the dendrimer architecture. Since the BC arms are derived from oligomeric segments, they are referred to as ‘polymeric dendrimers’51. These more flexible and extended structures exhibit different properties compared with the more compact traditional dendrimers. Fréchet, Hawker, and coworkers52 have utilized living polymerization and a staged polymerization process in which latent polymerization sites are incorporated within growing chains to produce dendrigrafts of mixed composition and narrow polydispersity.

Another exciting development is the emerging role that dendritic architecture is playing in the production of commodity polymers. A recent report by Guan et al.53 has shown that ethylene polymerizes to dendrigraft-polyethylene at low pressures, while high-pressure conditions produce only branched topologies. This occurs when using late-transitionmetal or Brookhart catalysts. Furthermore, these authors state that small amounts of dendrigraft-polyethylene architecture may be expected from analogous earlytransition- metal metallocene catalysts.

Dendrons and dendrimers
Dendrons and dendrimers are the most intensely investigated subsets of dendritic polymers. In the past decade, over 6000 papers have appeared dealing with this unique class of structure-controlled polymer. The word dendrimer is derived from the Greek words dendri- (tree-branch-like) and meros (part of), and was coined by Tomalia et al. about 20 years ago in the first full paper on poly (amidoamine) (PAMAM) dendrimers54. Since this early disclosure, over 100 dendrimer compositions (families) and 1000 dendrimer surface modifications have been reported. 

The two most widely studied dendrimer families are the Fréchet-type polyether compositions and the Tomalia-type PAMAM dendrimers. PAMAM dendrimers constitute the first dendrimer family to be commercialized, and represent the most extensively characterized and best-understood series at this time6,7.In view of the vast amount of literature in this field, the remaining overview will focus on PAMAM dendrimers. The scope will be limited to a discussion of the critical properties and unique quantized nanomodule features that make these materials suitable for nanoscale syntheses and manipulations6.

Dendrimer synthesis:divergent and convergent methods
In contrast to traditional polymers, dendrimers are unique core-shell structures possessing three basic architectural components (Fig. 4): a core (I), an interior of shells (generations) consisting of repeating BC units (II), and terminal functional groups (the outer shell or periphery) (III). In general, dendrimer synthesis involves divergent or convergent hierarchical assembly strategies that require the construction components shown in Scheme 1. Within each of these major approaches, there may be variations in methodology for BC or dendron construction. Many of these issues, together with experimental procedures, have been reviewed elsewhere55-57. 

PAMAM dendrimers are synthesized by the divergent approach. This involves   in situ BC construction around a core in stepwise, iterative stages to produce mathematically defined core-shell structures. Typically, ethylenediamine (core multiplicity, Nc = 4), ammonia (Nc = 3), or cystamine (Nc = 4) may be used as cores and allowed to undergo iterative, two-step reaction sequences. These sequences consist of: (a) an exhaustive alkylation of primary amines (Michael addition) with methyl acrylate; and (b) amidation of amplified ester groups with a large excess of ethylenediamine to produce primary amine terminal groups (Scheme 2). 

This first reaction sequence on the exposed core creates generation G = 0 (i.e. the core BC), where the number of arms (i.e. dendrons) anchored to the core is determined by Nc. Iteration of the alkylation-amidation sequence produces an amplification of terminal groups from one to two with the in situ creation of a BC at the anchoring site of the dendron constituting G = 1. Repeating these iterative sequences (Scheme 2) produces additional shells or generations of BCs that amplify mass and terminal groups according to the mathematical expressions described in Fig. 4, box section. It is apparent that both the core multiplicity (Nc) and BC multiplicity (Nb) determine the precise number of terminal groups (Z) and mass amplification as a function of generation (G). One may view these generation sequences as ‘quantized polymerization’ events. The assembly of reactive monomers25,26,58, BCs7,12,59,62, or dendrons7,60,61 aroundatomic or molecular cores to produce dendrimers according to divergent or convergent dendritic branching principles has been well demonstrated. Such systematic filling of molecular space around cores with BCs as a function of generational growth stages (BC shells) to give discrete, quantized bundles of nanoscale mass has been shown to be mathematically predictable62- 

Predicted molecular weights have been confirmed by mass spectrometry65-68 and other analytical methods12,60,69,70. Predicted numbers of BCs, Zs, and molecular weights as a function of G for a cystamine-core (Nc = 4) PAMAM dendrimer are shown in Fig. 4. It should be noted that molecular weights approximately double as one progresses from one generation to the next. The Zs and BCs amplify mathematically according to a power function, thus producing discrete, monodisperse structures with precise molecular weights and a nanoscale diameter enhancement (Fig. 4). however, withdivergent dendrimers, minor mass defects are often observed for higher generations as congestion-induced de Gennes dense packing begins to take effect12,71. 

Scheme 1 Hierarchical assembly scheme illustrating the options for constructing  dendrimers by either divergent or convergent synthetic strategies.

Scheme 2 Divergent synthesis of cystamine-dendri-PAMAM dendrimers utilizing the iterative sequence, (a) alkylation with methyl acrylate, followed by (b) amidation with excess ethylenediamine, to produce G = 3-7.
Unique dendrimer properties

Nanoscale monodispersity
The monodisperse nature of dendrimers has been verified extensively by mass spectrometry62,75, size-exclusion chromatography (SEC), gel electrophoresis70, and transmission electron microscopy (TEM)72. Comparison of a traditional, linear polymer size distribution Mw/Mn = 2-10 with PAMAM dendrimers (G = 1-7) is illustrated by SEC (Fig. 5). 

The respective dendrimer generations, as illustrated by TEM images for a G = 5-10 series of PAMAM dendrimers, are also displayed (Fig. 5)72. As is often the case, the level of monodispersity is determined by the skill of the synthetic chemist as well as the isolation and purification methods used. In general, convergent methods produce the most nearly monodisperse dendrimers, as determined by mass spectrometry. 

This is because the convergent growth process allows purification at each step of the synthesis and eliminates cumulative effects because of failed couplings7. Appropriately purified, convergently produced dendrimers are probably the most precise synthetic macromolecules that exist today. 

Mass spectrometry has shown that PAMAM dendrimers produced by the divergent method are remarkably monodisperse and have masses consistent with predicted values for earlier generations (G = 0-5) (Fig. 4)63,64,68. Even for higher generations, as one enters the de Gennes densely packed region, the molecular weight distributions remain very narrow (Mw/Mn = 1.05) and consistent, in spite of the fact that experimental masses deviate substantially from predicted theoretical values. Presumably, de Gennes dense packing produces a very regular and dependable effect that is manifested in the narrow molecular weight distributions4,71.

Fig. 5 TEM images of ethylenediamine core PAMAM dendrimers (G = 5-10) with their respective SEC traces (G = 0-9) illustrating their monodispersity.]

Nanoscale container and scaffolding properties 
Unimolecular container and scaffolding behavior appear to be periodic properties that are specific to each dendrimer family or series. These properties are determined by the size, shape, and multiplicity of the construction components that are used for the core, interior, and surface of the dendrimer (Fig. 4)4. Higher multiplicity components and those that contribute to ‘tethered congestion’ will hasten the development of container properties and rigid-surface scaffolding as a function of generation. Within the PAMAM dendrimer family, these periodic properties are generally manifested in three phases as shown in Fig. 6. 

The earlier generations (G = 0-3) do not exhibit any well-defined interior characteristics, whereas interior development related to geometric closure is observed for the intermediate generations (G = 4-7). Accessibility and departure from the interior is determined by the size and gating properties of the surface groups. At higher generations (G > 7), where de Gennes dense packing is severe, rigid-scaffolding properties are observed, allowing relatively little access to the interior except for very small guest molecules. 

The siteisolation and encapsulation properties of dendrimers have been reviewed recently by Esfand and Tomalia73, Hecht and Fréchet11, and Weener et al.74

Fig. 6 Periodic properties of PAMAM dendrimers as a function of generation. Various chemophysical dendrimer surfaces amplified according to Z = NcNb G. (Reproduced with permission from6. © 2004 Sigma-Aldrich Co.)

Amplification and functionalization of surface groups Dendrimers within a generational series can be expected to present their terminal groups in at least three different modes, namely as flexible, semiflexible, or rigid functionalized scaffolding (Fig. 6). 

Based on mathematically defined dendritic branching rules (Z = NcNb G), the various surface presentations become more congested and rigid as a function of increasing generation level. It is implicit that this surface amplification can be designed to control gating properties associated with unimolecular-container development. 

Furthermore, dendrimers may be viewed as versatile, nanosized objects that can be surface-functionalized with a vast array of chemical and application features (Fig. 7). The ability to control and engineer these parameters provides endless possibilities for use as modules in nanodevice design6,64,75-78. Recent reviews have begun to focus on this area11,58,77-81.

Nanoscale dimensions and shapes that mimic proteins In view of the extraordinary structure control and nanoscale dimensions observed for dendrimers, it is not surprising to find extensive interest in their use as globular protein mimics (Fig. 8)4,82. Based on their systematic size-scaling properties and electrophoretic and hydrodynamic behavior69,70, they are referred to as artificial proteins4,73,75. Substantial effort has focused recently on the use of dendrimers for site-isolation mimicry of proteins11,12, enzyme-like catalysis83, other biomimetic applications75,84, drug delivery73,84, surface engineering85, and light harvesting86,87. 

These fundamental properties have, in fact, led to their commercial use as globular protein replacements for gene therapy88,89, immuno diagnostics90,91, and a variety of other biological applications. Interestingly, properties optimized for dendrimer applications have been found to be size (generation)-specific as indicated in Fig. 8, and have been reviewed elsewhere4. 

Fig. 7 Options for modifying amine-terminated dendrimers using classical subnanoscale and nanoscale reagents. (Reproduced with permission from6. © 2004 Sigma-Aldrich Co.)

Fig. 8 Comparison of nanoscale protein dimensions with NH3-core PAMAM dendrimers (G = 1-7) and generational specific applications.

Dendrimer features of interest to nanoscientists
Nanosynthesis, self-assembly, and manipulation with dendrimers Dendrimers may be viewed as unique, information-processing nanoscale devices. Each architectural component manifests a specific function, while at the same time defining properties for nanostructures as they are grown generation by generation.

For example, the core may be thought of as the molecular information center from which size, shape, directionality, and multiplicity are expressed via covalent connectivity to the outer shells. Within the interior, one finds the BC amplification region, which defines the type and volume of interior void space that may be enclosed by the terminal groups as the dendrimer is grown. BC multiplicity (Nb) determines the density and degree of amplification as an exponential function of generation (G). The interior composition and volume of solvent-filled void space determines the extent and nature of the guest-host (endo-receptor) properties that are possible within a particular dendrimer family and generation. 

Finally, the surface consists of reactive or passive terminal groups that may perform several functions. With appropriate function-alization, they serve as a ‘template polymerization region’ as each generation is amplified and covalently attached to the precursor generation. The surface groups may also function as passive or reactive gates controlling entry or departure of guest molecules from the dendrimer interior.
These three architectural components (core, interior, and surface) essentially determine the physical and chemical properties, as well as the overall size, shape, and flexibility of a dendrimer. It is important to note that dendrimer diameters increase linearly as a function of shells or generations added, while the terminal functional groups increase exponentially as a function of generation. 

This dilemma enhances the ‘tethered congestion’ of the anchored dendrons as a function of generation because of the steric crowding of the end groups. As a consequence, lower generations are generally open, floppy structures, while higher generations become robust, less deformable spheroids, ellipsoids, or cylinders, depending on the shape and directionality of the core (Fig. 6).

In view of their precise, quantized nanoscale features, both dendrons and dendrimers have been used extensively in a variety of nanosynthesis and nanomanipulation operations. A small sampling of these activities includes the decorating of linear polymer backbones with dendrons to produce so-called linear-dendritic architectural copolymers or ‘dendronized polymers', a term coined by Schluter et al.92. Such dendronized polymers, if advanced to a sufficiently congested generational level, have exhibited rigid rod (dendrimeric nanotube) topologies reminiscent of carbon nanotubes when analyzed by TEM93,94. 

This area has been researched extensively by Schluter and others95, and these prototypes have been used in a variety of nanomani-pulations including the atomic force microscopy (AFM) study shown in Fig. 9a. Dendrons possessing thiol-functionalized focal points have been used to dendronize both Au nanoparticles and CdSe/CdS core-shell quantum dots by ligand-exchange of the protective surfactants used for their synthesis 96-98 (Fig. 9b). 
The ‘self assembly’ of functionalized dendrons on these nanoparticle surfaces leads to new ‘nanometal core-dendron shell megamers’. Such dendronizations yield passified nanometal clusters possessing innumerable chemical functionality options suitable for a wide variety of applications.

A related nanosynthesis involves the encapsulation of various metal salts within the interiors of PAMAM dendrimers (Fig. 9c). The dendrimers function as nanoreactors or containers, wherein the encapsulated salts are reduced to their respective zero valence metal states to produce a new class of ‘subnanoscale quantum dots’99

In some instances, these metal/dendrimer nanocomposites were electrostatically assembled ‘layer by layer’ to produce uniform multilayered thin films with the potential for tunable optical, electronic, or catalytic properties102. Recent work by  Zheng et al.103 has shown that variations of these prototypes (i.e. few-atom Au quantum dots) are highly fluorescent and water soluble104. They behave as multielectron artificial atoms with discrete, size-tunable electronic transitions throughout the visible and near-infrared region. It has been proposed that these constructs provide the ‘missing link’ between atomic and nanoparticle behavior in noble metals, and may open new opportunities for biological labels, energytransfer pairs, and light-emitting sources in nanoscale optoelectronics.
Fig. 9 Examples of (a) nanomanipulating dendronized linear polymers; (b) nanosynthesis of metal core-dendron shell megamers; and (c) nanosynthesis of subnanoscale quantum dots by metal encapsulation within a PAMAM dendrimer. (Part (a) reproduced with permission from95. © 2003 Wiley-VCH.)

Nanostructure control beyond the dendrimer
Dendrimer synthesis strategies now provide virtual control of macromolecular nanostructures as a function of size72,105, shape82,93, and surface or interior functional groups12. These strategies involve the covalent assembly of hierarchical components such as reactive monomers (A)26, BCs (B)7,59,62, and dendrons (C)61 around atomic or molecular cores according to divergent or convergent dendritic branching principles (Fig. 10)7,62,106. 

Systematic filling of the space around a core with shells of BCs (i.e. generations) produces discrete core-shell dendrimer structures (D). Dendrimers are quantized bundles of mass that possess amplified surface functionality and are mathematically predictable64. Predicted molecular weights and surface stoichiometry have been confirmed experimentally by mass spectrometry65,66, gel electrophoresis69,70, and other analytical methods72,105. 

It is now recognized that empirical structures such as A, B, and C may be used to define these hierarchical constructions. Such synthetic strategies have produced dendrimers with dimensions that extend well into the lower nanoscale region (1-20 nm)107. The precise structure control and unique new properties exhibited by these dendrimeric architectures have yielded many interesting advanced material properties11,108,109. 

Nanoscale dendrimeric containers100,108,110 and scaffolding12,81 have been used to template zero-valent-metal nanodomains79,100, nanoscale magnets111-113, electronconducting matrices114,115, and provide a variety of novel optoelectronic properties116,117. However, the use of such strategies for the synthesis of precise nanostructures (dendrons (C) and dendrimers (D)) larger than 15-20 nm has several serious disadvantages. Firstly, it is hampered by the large number of iterative synthetic steps required to attainhigher dimensions (e.g. a G = 9 PAMAM dendrimer with a diameter of ~10 nm requires 18 reaction steps). 

Secondly, these constructions are limited by the de Gennes densepacking phenomenon, which precludes ideal dendritic construction beyond certain limiting generations62,118. For these reasons, our attention has turned to the use of dendrimers as reactive modules for the rapid construction of controlled nanoarchitectures possessing a higher complexity  and dimensions beyond the dendrimer. We refer to these generic poly(dendrimers) as ‘megamers’ (E) (Fig. 10)23. Both randomly assembled megamers23, as well as structurecontrolled megamers23,119,120, have been demonstrated. Recently, new mathematically defined megamers (dendrimer clusters) or core-shell tecto(dendrimers) have been reported109,119- 

The principles of these structurecontrolled megamer syntheses mimic those used for the core-shell construction of dendrimers.  First, a megamer core reagent (usually a spheroid) is selected. Next, a limited amount of this reactive core reagent is combined with an excess of a megamer shell reagent. The objective is to completely saturate the target spheroid core surface with covalently bonded spheroidal shell reagent. Since the diameters of the megamer core and shell reagents are very well defined, it is possible to predict mathematically the number of megamer shell molecules required to saturate a targeted core dendrimer122.
These core-shell relationships have been analyzed mathematically as a function of the ratio of core (r1) and shell (r2) radii122. At low r1/r2 values (0.1-1.2), very important symmetry properties emerge as shown in Fig. 11. It can be seen that, when the core reagent is small and the shell reagent is larger, only a very limited number of shell dendrimers can be attached to the core dendrimer based on available space. However, when r1/r2 ≥1.2, the space becomes available to attach many more spheroidal shell reagents up to a discrete saturation level. The saturation number (Nmax) is well defined and can be predicted from the Mansfield-Tomalia-Rakesh equation (Fig. 

Fig. 10 Approximate nanoscale dimensions as a function of atoms, monomers, branch cells, dendrons, dendrimers, and megamers. (Reproduced with permission from6. © 2004 Sigma-Aldrich Co.)

Fig. 11 (a) Symmetry properties of core-shell structures, where r1/r2 < 1.2. (b) Sterically induced stoichiometry based on the respective radii of core and shell dendrimers. (c) Mansfield-Tomalia-Rakesh equation for calculating maximum shell filling when r1/r2 > 1.2. (Reproduced with permission from6. © 2004 Sigma-Aldrich Co.)

Synthesis of megamers
Saturated-shell-architecture approach
The general chemistry used in this approach involves the combination of a limited amount of an amine-terminated dendrimeric core reagent (e.g. a G = 5-7, NH2-terminated PAMAM dendrimer) with an excess of a carboxylic acid terminated dendrimeric shell reagent (e.g. PAMAM)120. 
These two charge-differentiated species are allowed to self assemble into an electrostatically driven, supramolecular, core-shell tecto(dendrimer) architecture. After equilibration, covalent-bond formation at these charge-neutralized dendrimer contact sites is induced with carbodiimide reagents (Fig. 12a)120,121.

The carboxylic acid terminated shell-reagent dendrimers (e.g. G = 3 or 5) are synthesized by ring opening of succinic anhydride with the appropriate amine-terminated PAMAM dendrimers. All reactions leading to core-shell tecto(dendrimers) are performed in the presence of LiCl at room temperature as dilute solutions (~0.5 wt.%) in water. Equilibration times of 16-20 hours are required to complete the charge-neutralized self assembly of excess shell reagent around the limited core dendrimer reagent. 

Following this self assembly and equilibration, a linking reagent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, is added to bond the assembly of dendrimeric shell reagents covalently to a single dendrimeric core reagent at the amine-carboxylic acid interaction sites. These sites are presumed to reside primarily at the exterior of the core dendrimer reagent23,120. 

Remarkably, monodisperse products are obtained by performing the core-shell self-assembly reactions in the presence of LiCl. In the absence of LiCl, these reactions yield bimodal or trimodal product mass distributions as observed by SEC, and are presumed to be the result of clustering of the amine-terminated core reagent into various domain sizes. Such clustering of amine-terminated PAMAM dendrimers has been noted in earlier work72. 

Attempts to charge neutralize these polydisperse domains subsequently with anionic dendrimeric shell reagent produces a broad product distribution. Reversing the terminal functional groups on the core and shell reagents (i.e. using carboxylicacid- terminated PAMAM dendrimer as the core and excess amine-terminated PAMAM dendrimer as the shell reagent) under identical reaction conditions does not yield the desired product. The reason for this is not evident from our studies so far.

Unsaturated-shell-architecture approach
The second approach, the direct covalent-bond-formation method, produces semicontrolled, partially filled shell structures23,75. It involves the reaction of a limited amount of a nucleophilic dendrimeric core reagent with an excess of an electrophilic dendrimeric shell reagent, Fig. 12b121. This route involves the random parking of the reactive shell reagent on a core-substrate surface. As a consequence, partially filled shell products are obtained, which possess relatively narrow but not precise molecular weight distributions, as noted for saturated core-shell architectures120. These distributions are determined by the core-shell parking efficiency prior to covalent bond formation. Various PAMAM dendrimeric core reagents (either amineor ester-functionalized) are each allowed to react with an excess of an appropriate PAMAM dendrimeric shell reagent. The reactions are performed at 40°C in methanol and monitored by Fourier transform infrared spectroscopy,

13C nuclear magnetic resonance, SEC, and gel electrophoresis. Conversions in step A (Fig. 12b) are monitored by SEC and confirmed by observing the formation of shorter retention time products, consistent with higher molecular weight structures. 

Additional evidence can be gained by observing the loss of the migratory band associated with the dendrimeric core reagent present in the initial reaction mixture, accompanied by the formation of a higher molecular weight product, which displays a much shorter migratory band position on the electrophoretic gel. In fact, the molecular weights of the resulting core-shell tecto(dendrimers) can be estimated by comparing the migratory time of the core shell products with the migration distances of the PAMAM dendrimer reagents (e.g. G = 2-10) used for their construction69,70.

It is important to perform capping reactions on the surface of the resulting unsaturated, ester-terminated core-shell products in order to pacify the highly reactive amine cleft surfaces against further reaction. Preferred capping reagents for pacifying the ester domains of the surface are either 2-aminoethanol or tris (hydroxymethyl) aminomethane75.

12 (a) Saturated-shell-architecture approach to megamer synthesis. All surface dendrimers are carboxylic acid terminated. 

(b) Step A: unsaturated-shell-architectureapproach to megamer synthesis. Step B: surface-capping reactions.

Reactivity of dendrimers
Dendritic species possessing an unsaturated outer monomer shell of ester and amine domains exhibit autoreactive behavior. They are often encountered if a completely saturated state of either ester or amine groups is not attained. These species, which include missing-branch structures, lead to the formation of monodendrimers containing macrocyclic terminal groups as well as moderate amounts of megamers (i.e. dimeric, trimeric species, etc.). 

Ideal dendrimer structures (i.e. saturated outer-monomershell products) can, however, be separated from these side products by silica gel column chromatography and preparative thin-layer chromatography isolation techniques.

Ideal dendrimer structures that exhibit mathematically predictable masses, as well as unsaturated monomer-shell products exhibiting mass defects, are readily characterized by electrospray and matrix-assisted laser desorption/ionizationtime of flight mass spectrometry65-68.

Recently, we reported additional evidence that unfilled outer-monomer-shell species are indeed autoreactive intermediates that do lead to megamer formation. In general, saturated-shell PAMAM dendrimers (i.e. all-amine- or all- ester-group-saturated surfaces) are very robust species (analogous to inert gas configurations observed at the atomic level). In this regard, they do not exhibit autoreactive characteristics. Such samples may be stored for months or years without change. On the other hand, PAMAM dendrimer samples possessing unfilled monomer shells (amine and ester group domains on the dendrimer surface) are notorious for exhibiting autoreactive properties leading to terminal looping (macrocycle formation) and megamer formation63,64. 
Remarkably, these autoreactivity patterns are also observed for the dimensionally larger core-shell tecto(dendrimer) architectures. For example, saturated-shell core-shell tecto(dendrimer) architectures exhibit no autoreactivity120; whereas partially filled shell, core-shell tecto(dendrimers) exhibit profound autoreactivity75, unless pacified by reagents possessing orthogonally reactive functionalities. This behavior is comparable to the modular reactivity patterns of atoms and basic dendrimers as illustrated in (Fig. 13)6,75,121.

Fig. 13 Quantized module (building block) reactivity patterns at the subnanoscale (atoms), lower nanoscale (dendrimers), and higher nanoscale (core-shell tecto(dendrimers)) levels, involving unsaturated electron, monomer, or dendrimer shells, respectively. (Reproduced with permission from6. © 2004 Sigma-Aldrich Co.)

Dendritic polymers are expected to play a key role as enabling building blocks for nanotechnology6 during the  21st century, just as the three traditional architectural classes of synthetic polymers have successfully fulfilled critical material and functional needs over the past half-century. 

Thecontrolled shape, size, and differentiated functionality of dendrimers; their ability to provide both isotropic and anisotropic assemblies; their compatibility with many other nanoscale building blocks such as DNA, metal nanocrystals, and nanotubes; their potential for ordered self-assembly; their ability to combine both organic and inorganic components; and their propensity to either encapsulate or be engineered into unimolecular functional devices make dendrimers uniquely versatile among existing nanoscale building blocks and materials. 

Dendritic polymers, especially dendrons and dendrimers, are expected to fulfill an important role as fundamental modules for nanoscale synthesis. It is from this perspective that it is appropriate to be optimistic about the future of this new major polymer class, the dendritic state. MT


3. Dendrimers as biomimetic artificial proteins
Based on their dimensional length scaling, narrow size distribution, and other biomimetic properties, dendrimers are often referred to as bartificial proteinsQ [24–28]. Within the PAMAM family, they closely match the sizes and contours of many important proteins and bioassemblies. 

For example, insulin (3 nm), cytochrome C (4 nm), and hemoglobin (5.5 nm) are approximately the same size and shape as ammonia-core PAMAM dendrimers generations 3, 4 and 5, respectively. 

Furthermore, generations 5 and 6 PAMAM dendrimers have diameters approximately equivalent to the thickness of lipid bilayer membranes (~5.5 nm) of biological cells, while a generation 2 dendrimer matches the width (2.4 nm) of DNA duplexes. 

These duplexes form stable complexes with histone clusters to condense and store DNAwithin the nucleosome of cells. Undoubtedly, the close match in size and shape between histone clusters and PAMAM dendrimers of generations 7–10 accounts for the extraordinary stability of DNA–PAMAM complexes, as well as the enhanced gene expression observed for these dhistone mimicsT compared with lower generation (G=1–5) PAMAM dendrimers [29–31]. 
Although there are many similarities between dendrimers and globular proteins, it is also important to note significant differences. Whereas globular proteins are tertiary structures resulting from the intricate folding of sequenced linear structures, they are extremely fragile and susceptible to denaturing conditions, such as temperature, light, and pH. 

Furthermore, folded proteins generally produce densely packed interiors and surfaces possessing highly heterogeneous domains of functionality, hydrophobicity, and hydrophilicity. 

By contrast, dendrimers are known to be robust, covalently fixed, three dimensional structures possessing both a solvent-filled interior core (nanoscale container) as well as a homogenous, mathematically defined, exterior surface functionality (nano-scaffold).

4. Dendrimers as nanoscale containers
Dendrimers may be visualized as consisting of three critical architectural domains:
(i) the multivalent surface, containing a larger number of potentially reactive/passive sites (nano-scaffolding), 
(ii) the interior shells (i.e.,branch cell layers defined by dendrons) surrou-nding the core, and 
(iii) the core to which the dendrons are attached. 
The two latter domains represent well-defined nano-environments, which are protected from the outside by the dendrimer surface (nanoscale containers) in the case of higher generation dendrimers. 

These three domains can be tailored for a specific purpose, i.e., to function as a dendritic sensor, drug carrier, or as a drug. The high density of exo-presented surface functionalities makes the dendritic surface well-suited as a nano-scaffold where the close proximity of functional groups is important (polyvalency) or for receptor mediated targeting purposes. 

On the other hand, the interior is well-suited for host–guest interaction and encapsulation of guest molecules. Tomalia demonstrated by electron microscopy observation that sodium carboxylated PAMAM dendrimers possess topologies reminiscent of regular classical micelles [4]. 

It was also noted from electron micrographs that a large population of individual dendrimers possessed a hollow core. Supporting these observations, Turro and colleagues designed a hydrophobic 12-carbon atom alkylene chain into the core of a homologous series of PAMAM dendrimers (G=2, 3, and 4) to mimic the hydrophobic and hydrophilic core–shell topology of a regular micelle. 
The hosting properties of this series towards a hydrophobic dye as a guest molecule were then compared with a PAMAM dendrimer series possessing non-hydrophobic cores (e.g., NH3 and ethylenediamine). 
Dramatically enhanced emission of the hydrophobic dye was noted in aqueous solution in the presence of hydrophobic versus hydrophilic cored dendrimers [32]. The micelle-mimetic behavior of dendrimers was also observed in recent molecular dynamic studies. Depending on the conditions of the bulk solution, i.e., its polarity, ionic strength and pH, dendrimers adopt conformations of different shape and density. 

For example, poly(propylene imine) (PPI) and PAMAM dendrimers with primary amines as surface groups exhibit extended conformations upon lowering the pH due to electrostatic repulsion between protonated tertiary amines in the interior as well as between surface primary amines, thus forcing the dendritic branches apart. At pHN9, back-folding occurs as a consequence of hydrogen bonding between interior protonated tertiary amines and surface primary amines, resulting in a denser interior [33,34]. 

Of course, these pH-related conformational changes are dependent on the charge of the respective functional groups. For PPI dendrimers having surface carboxylate groups instead of amines, small angle neutron scattering (SANS) and NMR measurements show that these dendrimers possess the most extended conformations at pH 4 and 

This observation has been related to electrostatic repulsion between interior cationic protonated tertiary amines at low pH, and electrostatic repulsion between anionic deprotonated carboxylates on the surface at high pH. Either positive or negative repulsions are forcing the dendritic branches apart. At pH 6, carboxylate-terminated PPI dendrimers exhibit no net charge, resulting in a tighter conformation controlled by intramolecular hydrogen bonds between terminal groups and groups in the core [35]. In addition to the pH of the bulk solution, the solvent polarity will effect the dendrimer conformation in a similar way as observed for micelles. 

Recent NMR studies performed on polar PPI dendrimers indicate that apolar solvents such as benzene will favor intramolecular interactions, i.e., hydrogen bonds, resulting in back-folding of the dendrimer arms into the interior, whereas more polar solvents such as chloroform will compete for and thus weaken these intramolecular interactions in favor of solvated arms, allowing a more extended conformation of the dendrimers [36]. 

As a result of these interactions, polar dendrimers will have a higher density at the core in poor (apolar) solvents and a higher density at the surface in good (polar) solvents [37]. These solventdependent conformational changes will reverse in the case of less polar dendrimers (i.e., dendrimers containing aryl groups or other hydrophobic moieties as building elements), thus forcing these dendrimers to behave as inverse micelles [38]. 

A critical property difference relative to micelles is the increased density of surface groups with higher generations. At some generational level, the surface groups will reach the so-called bde Gennes dense packing limit and seal the interior from the bulk solution (Fig. 4) [39,40]. 

This limit depends on the strength of intramolecular interactions between adjacent surface groups and, therefore, on the condition of the bulk solution (i.e., pH, polarity, temperature). This feature can be utilized to tailor the encapsulation and release properties of dendrimers, for example in drug delivery applications, coined by Meijer and colleagues as the ddendritic box approach [40]. 

This phenomenon was originally noted for PAMAM dendrimers by Tomalia and co-workers and referred to as unimolecular encapsulation [41,42]. Surface-modification of G=5 PPI dendrimers with Boc-protected amino acids induced dendrimer encapsulation properties by the formation of dense, hydrogen-bonded surface shells with solid-state character. Small guest-molecules were captured in such dendrimer interiors and were unable to escape even after extensive dialysis. 

The maximum amount of entrapped guest molecules was directly proportional to the shape and size of the guest molecules, as well as to the amount, shape and size of the available internal dendrimer cavities. Four large guest-molecules (i.e., Rose Bengal) and 8–10 small guest-molecules (i.e., pnitrobenzoic acid) could be simultane-ously encapsulated within PPI dendrimers containing four large and twelve smaller cavities. 

Remarkably, this dendrimer box could be opened under controlled conditions to release either some or all of the entrapped guest molecules. For example, partial hydrolysis of the hydrogen-bonded Boc-shell liberated only small guest-molecules, whereas total hydrolysis released all sizes of entrapped molecules [40,43,44]. 

Although the ddendritic boxT concept demonstrates the unique shape-dependent cargo space that can be found in certain dendrimers, it does not offer a practical means for delivering and releasing therapeutic drugs under physiological conditions.

A major objective of our current effort at Dendritic Nano- Technologies Inc. has been the resolution of these problems. The robust covalent structural features of dendrimers offer a variety of defined sizes and shapes (i.e., cargo spaces) and permit extensive dendrimer surface modifications to effectively control guestmolecule entry and exit parameters. 

From a thermodynamic perspective, free guest-molecules (i.e., drugs) can be distinguished from those encapsulated or bound in a complex by finite energy barriers related to the ease of entry and departure to dendrimer cavities. If the drug molecule is significantly large or incompatible with either the dimension or hydrophilic/lipophilic character of the dendrimer cavity, a complex might not form, or the guest might only be partially encapsulated within the dendrimer host. 

A hydrophobic drug would be expected to associate with a dendrimer core to achieve maximum contact with its hydrophobic domain. In addition, the hydrophobic character of this guest molecule would be expected to isolate itself from the dendrimer surface and the interface to the bulk solution to afford minimum contact with polar and aqueous domains (i.e., physiological media).

Notably, the hydrophobic and hydrophilic properties, as well as other non-covalent binding properties of these spatial binding-sites are expected to strongly influence these guest–host relationships. 

Analysis of a typical symmetrically branched dendrimer makes it apparent that there are other subtle, and yet important, parameters that could control the interior space of a dendrimer and influence guest–host interactions. These include components such as branching angles, rotational angles, and the length of a repeat-unit segment [45]. 

Of equal importance are the properties of the core. Within a homologous PAMAM dendrimer series, the effect of changing the length scale of the core on dendrimer guest–host properties was studied. Specifically, a series of polyhydroxy-surfaced PAMAM dendrimers with core molecules differing in length by one carbon atom (NH2–Cn–NH2 with n =2–6) were synthesized. 

Three aromatic carboxylic acids, differing systematically by one aromatic ring (benzoic acid, 1-naphthoic acid, 9-anthracene carboxylic acid), were examined as guest-molecule probes. Two sets of dendrimers, possessing 24 and 48 surface hydroxy groups, were investigated [46]. 

The observed trends can be summarized as follows:
(i) Ingeneral, all dendritic hosts accommodated larger amounts of the smaller guest-molecule (i.e., molar uptake benzoicN1-naphthoicN9-anthracene carboxylic acid). This observation was particularly significant for the more congested dendrimer surface having 48 surface OH-groups. 

(ii) Uptake maxima values specific to both the core size and the specific guest-probe were noted. This observation might be related to the combination of shape and lipophilicity manifested by the guest probe. 

(iii) A decrease in the molar uptake was measured for all probes as the core
was enhanced beyond an ideal dimension (i.e., 5–6 carbons). It is, therefore, obvious that both core size and surface congestion dramatically affect the cargospace of the dendrimer host. 
Furthermore, it is apparent that size and shape of the guest probe can significantly affect the maximum loading as a function of core size. Finally, it should also be noted that for the dendrimers G=2 (24-OH) and G=3 (48-OH), the guest probes had desirable release properties from the host as a function of time, when re-dissolved in water. 

Performing these same experiments using a dendrimer with more densely packed surface groups (i.e., G=4 with 96 surface OHgroups) appeared to produce dendritic box behavior. Although guest molecules could be encapsulated within the core, the release from the host was delayed as determined by analysis after extensive dialysis [46]. 

Structure–property relationships in dendritic encapsulation have been studied extensively mainly using photoactive and redox-active model dendrimers to gain a better understanding of the structural effects that cores and branches have on encapsulation [47–49].

Specific binding of guest molecules to the dendrimer core can affect the loading capacity by enhancing specific interactions between the core and guest, i.e., hydrophobic and polar interactions. Dendrimers specifically tailored to bind hydrophobic guests to the core have been created by Diederich and coworkers and coined ddendrophanes. These water-soluble dendrophanes are built around a cyclophane core, and can bind aromatic compounds, presumably via k–k interactions. 

Dendrophanes were shown to be excellent carriers of steroids [50,51]. The same group synthesized dendrimers tailored to bind more polar bioactive compounds to the core, coined ddendrocleftsT [52,53]. 

In another approach, the surface amines of PAMAM dendrimers were modified with tris(hydroxymethyl)aminomethane (TRIS) to create water-soluble dendrimers capable of binding carboxylic aromatic, antibacterial compounds, which could be released by lowering the pH 

An alternative approach to creating dendritic hosts with highly selective guest recognition utilized the principle of bmolecular imprintingT [54]. 

A dendrimer consisting of a porphyrin core and a surface containing terminal double bonds was polymerized into a polydendritic network. Subsequently, the base-labile ester bonds between cores and dendritic wedges were cleaved, releasing the porphyrin core from the dendritic polymer. 

This polymer was capable of selectively binding porphyrins with association constants of 1.4_105 M_1. Very recently, an impressive approach has been presented, using tandem mass spectrometry, i.e., the combination of electrospray ionization (ESI) and collision-induced dissociation (CID) mass spectrometers connected in series, to investigate the dynamic behavior of host–guest dendrimer complexes [55]. This approach offers the potential to provide better insights into these constructs.

Fig. 4. Periodic properties of PAMAM dendrimers generations G=0–10 depicting the distances between surface charges (Z–Z), including the dde Gennes dense packingT, the generation-dependent size and shape of dendrimers, and their dnanoscale-containerT and dnano-scaffoldingT properties.

5. Dendrimers in gene transfection
The use of dendrimers as gene transfection agents and drug delivery devices has been thoroughly reviewed very recently [24,56–58]. Therefore, only a few highlights are summarized together with additional recent studies. 

Dendrimers are very actively under investigation for the delivery of DNA and small organic molecule drugs, especially for cancer therapy. Numerous reports have been published describing the use of amino-terminated PAMAM or PPI dendrimers as non-viral gene transfer agents, enhancing the transfection of DNA by endocytosis and, ultimately, into the cell nucleus [30,59–68]. 

It should be noted that dendrimers of high structural flexibility and partially degraded high-generation dendrimers (i.e., hyperbranched architectures) appear to be better suited for certain gene delivery operations than intact high-generation symmetrical dendrimers. Perhaps this is due to their enhanced flexibility, which allows the formation of more compact complexes with DNA [69–71].

Furthermore, it has been found that maximum transfection efficiency is obtained with a net positive charge on the complexes (i.e., an excess of primary amines over DNA phosphates). 

To gain a better understanding of the rules that govern dendrimer-based gene delivery, a series of amphiphilic dendrimers based on the rigid diphenylethyne core was synthesized and their activity as transfection agents described. 

Drawing on low toxicity, geometric tunability, and ease of functionalization of cationic dendrimers on the one hand and classic self-assembly of amphiphilic molecules on the other, a set of molecular building blocks was developed to prepare amphiphilic dendrimers with rigid cores.

These dendrimers featured a variety of geometries and substitution patterns, all of which showed high transfection activity, but also brought a couple of surprises. A sharp maximum in the structure–activity relationship was observed, based on the hydrophobic component of the dendrimer. The hydrophobic parameters influenced the DNA binding and transport more strongly than anticipated, exhibiting lower toxicity and an unusual serum effect. 

In contrast to classical cationic dendrimers, these dendrimers did not show a minimum size limitation for transfection. However, while an optimum molecular weight greater than 116 kDa was found for PAMAM dendrimers, these constructs gave an optimum activity with rather small dendrimers having molecular weights between 1500 and 2700 Da [72]. Conjugation of  a-, h-, and gcyclodextrins to the surface of PAMAM dendrimers improved the transfection efficiency as well,especially using a-cyclodextrin in a 2.4 :1 ratio covalently bound to the G = 3 dendrimer surface. 

The measured transfection efficiency was approximately 100-times higher for the dendrimer conjugate than for the dendrimer alone or of a physical mixture between dendrimer and a-cyclodextrin [73]. Similarly, the attachment of small hydrophobic molecules such as the fluorescent dye Oregon green 488 to the dendrimer surface produced a similar enhancement in transfection efficiency [74]. 

The physical mixing of linear anionic oligonucleotides (i.e.,sequence lengths between 6 and 55 bases) as well as hairpin conformations with plasmid DNA prior to the addition of either a commercially available PAMAM dendrimerbased product (SuperFectR) or generations 3–5 phosphorus dendrimers was also examined in an attempt to improve the transfection efficiency. While the efficiency increased with the length of the oligonucleotide up to the 36-mer, the conformation of the oligomer was of less importance. 

The mixing with dextran sulfate having a similar molecular weight as the oligonucleotides had a similar effect, whereas, interestingly, the addition of non-ionic dextran having the same molecular weight had no effect upon plasmid expression.It was concluded that the presence of these charged macromolecules reduced the packing density of the dendrimer–DNA complex, and its enhanced flexibility resulted in improved transfection efficiency [75]. 

In another study, dendritic amidoamine side chains of different generations were covalently attached to the polysaccharide chitosan in an attempt to combine the biological activity of chitosan in gene delivery, antibacterial activity, and wound healing activity with the delivery benefits found for dendrimers. While several chitosan–dendrimer hybrids have been synthesized and characterized, their biological activity has still to be determined [76]. 

Amphiphilic, Tomalia-type PAMAM dendrons, generations 1 to 4, were synthesized utilizing di-n-dodecylamine as the core. It was anticipated that the hydrophobic component would mimic the membrane transfection ability of natural phospholipids such as dioleoylphosphatidylethanolamine (DOPE) and enhance membrane penetration. These constructs formed facile complexes with DNA and, in case of the G=2–4 dendrons, were able to cross cell membranes and efficiently deliver DNA [77].

Several recent studies combined dendrimers or dendritic structures with amino acids and peptides to improve the delivery ability of amino acid-based devices or to create more biocompatible molecules. 

Monodisperse, dendritic poly(l-lysine)s at several generational levels were prepared in order to compare the gene transfection properties against linear and branched poly(lysine) architectures. Poly(lysine) dendrimers possessing 64 and 128 surface amino groups exhibited efficient gene transfection properties in several cultivated cell lines without significant cytotoxicity [78]. 

Polycationic h-alanine-based dendrimers, generations 2 to 4, were synthesized and characterized. These molecules are expected to be efficient gene transfection agents due to their structural resemblance to natural biomolecules [79].

Surface functionalization of polyphenylene dendrimers with up to 16 lysine residues or short peptide sequences with up to 5 lysine or glutamic acid repeat units was carried out in order to utilize these compounds as models to study DNA complexation and condensation and as building blocks for novel supramolecular architectures [80]. 

Recently, chiral depsipeptide dendrimers were introduced, mimicking the structure of natural depsipetides, which consist of a-hydroxy and a-amino acids, connected by ester and amide linkages [81]. 

Chirality is a characteristic property of biomolecules and is crucial in the interplay between structure and function. This concept has been extended to depsipeptide dendrons based on tartaric acid as branching juncture and N-aminocapronic acid as spacer. 

The chirality of these compounds and the potential formation of a chiral secondary structure, mimicking biomolecules even closer found [82].

6. Dendrimers in drug delivery
In addition to DNA, dendrimers have been utilized to carry a variety of small molecule pharmaceuticals. Encapsulation of the well-known anticancer drug cisplatin within PAMAM dendrimers gave conjugates that exhibited slower release, higher accumulation in solid tumors, and lower toxicity compared to free cisplatin [83]. 

Similarly, the encapsulation of silver salts within PAMAM dendrimers produced conjugates exhibiting slow silver release rates and antimicrobial activity against various Gram positive bacteria [84,85]. 

In another study, PAMAM dendrimers with 4, 8, and 16 terminal ester groups were converted to hydroxy-terminated molecules using TRIS to reduce their potential cytotoxicity. These dendrimers were able to encapsulate small acidic molecules such as benzoic acid and 2,6-dibromo-4-nitrophenol in 1:1 and 2:1 (drug : dendrimer) ratios but did not form a complex with the non-acidic drug tioconazole. Presumably, the guest molecules were retained within the dendritic branching clefts by hydrogen bonding with interior protonated amide groups. 

Therefore, the inclusion complexes were observed to separate after deprotonation of these amide groups at pHb7 [86]. Two polyester-based dendrimers (generation 4 with trisphenolic core) were synthesized, one carrying a hydroxy surface, the other a tri(ethylene glycol) monomethyl ether surface. 

These dendrimers were compared to a 3-arm poly(ethylene oxide) star polymer, carrying G=2 dendritic polyester units at the surface. The star polymer gave the most promising results regarding cytotoxicity and systemic circulatory half-life (72 h). Therefore, the anticancer drug doxorubicin was covalently bound to this carrier via an acid-labile hydrazone linkage. The cytotoxicity of doxorubicin was significantly reduced (80–98%), and the drug was successfully taken up by several cancer cell lines [87]. 

In another study, poly(ethylene glycol) monomethyl ether chains with molecular weights of 550 and 2000 Da were attached via urethane bonds to generations 3 and 4 PAMAM dendrimers. The encapsulation behavior of these compounds for the anticancer drugs adriamycin and methotrexate was studied (Fig. 5). The highest encapsulation efficiency, with on average 6.5 adriamycin molecules and 26 methotrexate molecules per dendrimer, was found for the G=4 PAMAM terminated with PEG2000 chains. The drug release from this dendrimer was slow at low ionic strength but fast in isotonic solution [88]. 

The anticancer drug 5-fluorouracil encapsulated into G=4 PAMAM dendrimers with carboxymethyl PEG5000 surface chains revealed reasonable drug loading, and reduced release rate and hemolytic toxicity compared to the non-PEGylated dendrimer (Fig. 5) [89]. 

The anti-inflammatory drug ibuprofen was used as a model compound to study its complexation and encapsulation into generations 3 and 4 PAMAM dendrimers and a hyperbranched polyester having approximately 128 surface OH-groups. It was found that up to 78 ibuprofen molecules were complexed by the PAMAM dendrimers through electrostatic interactions between the dendrimer amines and the carboxyl group of the drug. In contrast, up to 24 drug molecules were encapsulated into the hyperbranched polyol. The drug was successfully transported into lung epithelial carcinoma cells by the dendrimers. [90]

Recent studies using Caco-2 cell lines have indicated that low generation PAMAM dendrimers cross cell membranes presumably through a combination of two processes, i.e., paracellular transport and adsorptive endocytosis, while cell efflux systems have a minor effect. [91]

The effect of PAMAM dendrimer generation size and surface functional group on the aqueous solubility, and therefore, bioavailability of nifedipine has been studied. The solubility enhancement of nifedipine was higher in the presence of ester-terminated dendrimers than their amino-terminated analogues possessing the same number of surface groups. Not unexpected, the nifedipine solubility increased with the size of the dendrimers .[92]

Furthermore, recent work has shown that PAMAM dendrimers enhanced the bioavailability of indomethacin in transdermal delivery applications [93]. Branched poly(l-glutamic acid) chains were centered around PAMAM dendrimers generations 2 and 3 and poly(ethylene imine) (PEI) cores to create new biodegradable polymers with improved biodistribution and targeting ability.

These constructs were surface-terminated with poly (ethylene glycol) chains to enhance their biocompatibility, and folic acid receptors to introduce cellspecific targeting. Cell binding studies have been performed using the epidermal carcinoma cell line, KB [94].

Star-shaped polylactide (PLA) was synthesized by bulk polymerization with a G=1 PAMAM dendrimer as initiator. Unlike linear PLA of similar molecular weight, the branched construct had higher hydrophilicity and faster degradation rate with significantly accelerated release of water-soluble bovine serum albumin as the model drug. These constructs are assumed to have potential in the delivery of hydrophilic drugs in tissue engineering, including growth factor and antibodies to induce tissue regeneration [95].

Two studies employed dendrimers as building blocks in the construction of new drug delivery devices. 

The first study extended the dendrimer-PEO motif by designing dbow-tieT structures, i.e., covalently connecting two polyester dendrons, where one dendron provides multiple functional handles for the attachment of drug molecules, while the other dendron is used for the attachment of solubilizing poly(ethylene oxide) chains. By varying the generations of dendrons and the mass of the PEO chains, molecular weight, architecture, and drug loading capacity can be readily controlled. A library of eight carriers with molecular weights between 20 and 160 kDa was synthesized and characterized [96]. 

The other study employed generation 4 PAMAM dendrimers to construct dendrimer/poly(styrenesulfonate) (PSS) microcapsules following a layer-by-layer deposition protocol of both constituents around a removable melamine formaldehyde colloidal core. These PAMAM/PSS capsules are expected to allow the selective encapsulation of drug into the capsule core and into the dendrimers, which are localized within the shell of the capsule, thus providing a dual release system of either two different drugs (i.e., drug cocktail) or of one drug released following two different time protocols (i.e., fast and sustained release). Doxorubicin hydrochloride has been used as a model compound for preliminary encapsulation and release studies [97].

Fig. 5. Schematic presentation of the encapsulation of anticancer drugs methotraxate (left) and 5-fluorouracil (right) into PEGylated generation 3 and 4 PAMAM dendrimers. (Adapted from Refs. [88,89].).

7. Dendrimers as imaging agents
Paramagnetic metal chelates such as Gd(III)- N,NV,NW,Nj-tetra-carboxymethyl-1,4,7,10-tetraazacyclododecane (Gd(III)-DOTA), Gd(III)-di-ethylenetriamine pentaacetic acid (Gd(III)-DTPA), and their derivatives increase the relaxation rate of surrounding water protons and are used as contrast agents for magnetic resonance imaging (MRI) [98,99]. 

The (Gd(III)-DTPA) conjugate is known commercially as MagnevistR (Schering AG) and is a widely used MRI contrast agent. However, shortcomings of these low molecular weight contrast agents are short circulation times within the body and inefficient discrimination between diseased and normal tissues. Subsequently, macromolecular Gd(III) complexes have been developed by conjugating Gd(III) chelates to biomedical polymers, including poly(amino acids), polysaccharides, and proteins to improve image contrast enhancement. 

These macromolecular agents have demonstrated superior contrast enhancement for blood pool imaging and cancer imaging in animal models. 

Unfortunately, the clinical application of macromolecular agents in general is limited by their slow excretion rate and, as a result, their accumulation within the body, i.e., the liver. In addition, the long residence time of MRI agents enhances the risk of potential toxicity by Gd(III) ions released during the metabolism of these agents [100,101]. In an attempt to facilitate the renal clearance of macromolecular Gd(III) complexes by blocking the renal tubular reabsorption, lysine has been co-injected with dendrimer- based macromolecular agents. However, it was found that this approach cannot facilitate the clearance of macromolecules of relatively high molecular weights [102].  

In another approach, the conjugation of (Gd(III)-DOTA) to poly(l-glutamic acid) (molecular weight 50 kDa) via the biodegradable disulfide spacer cystamine was studied to find a safe and effective macromolecular MRI contrast agent. Organic disulfide–thiol exchange reactions play a crucial role in biological systems, and the disulfide bond can be readily cleaved by thiols present in the body. 
The comparatively low (approximately 15 AM) thiol concentration in plasma results in a slow cleavage rate of the disulfide spacer and thus allows sufficient time for effective contrast-enhanced diagnostic imaging. The rate of disulfide cleavage and, subsequently, excretion of Gd(III) chelates after the MRI examination can be accelerated by administrating exogenous thiols [103,104].

In 2003, substantial attention was focused on the MRI field with the awarding of the Nobel Prize in Medicine to Prof. Paul C. Lauterbur, the father of the area [105]. As early as 1990, Lauterbur, Wiener and Tomalia pioneered the use of dendrimer-based MRI contrast agents by reporting some of the highest known relaxivities for these agents [106,107]. These extraordinary properties have been studied extensively in vivo during the last decade by Kobayashi and Brechbiel [108]. 

These properties appear to result from a combination of the geometrical amplification of chelated gadolinium that is possible on a dendrimer surface and higher rotational correlation times with minimal segmental motion that are intrinsic to these dendrimer conjugates. Consequently, dendrimer-based Gd(III) chelates consisting of generations 2 and 6 PAMAM dendrimers with 12 and 192 terminal surface amines conjugated to the chelating ligand 2- (4-isothiocyanatobenzyl)-6-methyldiethylenetriamine-pentaacetic acid through a thiourea linkage were synthesized and used in vivo with rabbits. 

These contrast agents exhibited excellent MRI images of blood vessels upon intravenous injection. The blood circulation times were sufficiently long, with more than 100 min for large dendrimer conjugates such as the G =6 PAMAM-TU-Gd(III)-DTPA [109]. 

The results were confirmed by studies employing generations 7, 9, and 10 PAMAM dendrimer-Gd(III) chelates containing up to 1860 Gd(III) ions per dendrimer. In this study, the total molecular relaxivity increased strongly with the molecular weight, although relaxivity saturation was observed [110]. 

A linear increase in the relaxivity as a function of molecular weight was also found in another study, utilizing generation 3 and 5 dendrimer polychelates. These dendrimer polychelates were exploited for high-quality MR angiography (MRA) images up to 60 min post injection. A less desired feature of these MRI dendrimers was the retention of up to 40% of the larger derivatives in the liver, which were present even seven days after administration. 

This problem could be addressed by incorporation of PEG-subunits into Gd(III)-chelated PAMAM dendrimers, reducing the liver retention to 1–8% after seven days [111]. 

The clearance time of dendrimer-based MRI agents and the potential risk of Gd(III) release were the subject of another study. Six small dendrimer-based agents were synthesized based on PAMAM and diaminobutane (DAB) dendrimers, and their pharmacokinetics, whole-body retention, and dynamic MRI were evaluated. DAB-based agents cleared more rapidly from the body than PAMAM dendrimer-based agents with the same number of surface groups. As a result of this study, one generation 2 PAMAM and two generations 2 and 3 DAB dendrimers were identified as contrast agents with potential for use in clinical applications [112].

Most interestingly, in a recent study it was found that the molecular size of a dendrimer-based MRI agent altered the route of excretion. Contrast agents with molecular weight less than 60 kDa were excreted through the kidney resulting in these agents being potentially suitable as functional renal contrast agents. 

Hydrophilic and larger-sized contrast agents were found better suited for use as blood pool contrast agents. Hydrophobic variants formed with PEI diaminobutane dendrimer cores created liver contrast agents. Larger hydrophilic agents were useful for lymphatic imaging. 

Finally, contrast agents conjugated to either monoclonal antibodies or avidin were able to function as tumor-specific contrasts. The potential of these dendrimer-based MRI agents has been recognized by the pharmaceutical industry and resulted in several commercial developments. 

For example, the 24-Gd(III)-DTPA cascade polymer and Gadomer 17 have been introduced. These compounds consist of trimesic acid cores connected to generation 2 poly (lysine) dendrons bearing 24 DTPA and 24 DOTA chelating groups, respectively [113,114]. Gadomer 17 is suitable for blood-pool imaging, similar to the known linear Gd(III)-DTPA-poly(lysine), but shows a superior elimination rate presumably as a result of the globular nature of the dendrimer derivative. 

At the same time, it has been found in several recent p’cokinetic studies of Gadomer 17 and related compounds that dendrimer-based contrast agentsextend the time window of dynamic contrast-enhanced MRI [115]. However, the internal rotational flexibility of the Gd(III) chelate in Gadomer 17 and the slow water exchange rate, both reducing proton relaxivity, are currently limiting factors in its applicability to MRI [116]. 

A very recent study describes a convenient methodology for the synthesis of Gd(III)- DTPA-terminated poly(propylene imine) dendrimers with tunable molecular relaxivities as contrast agent s [117]. 

An alternative approach is based on the utilization of dendrimers carrying four to twelve glucose moieties at the surface and Gd(III) ions coordinated to the core [118].

Another application of dendrimer-based diagnostics includes DNA-dendrimers, which are constructed by sequential hybridizations of partially complementary hetero-duplexes (dDNA monomersT). DNA dendrimers with up to two million terminal oligonucleotide strands have been reported. These DNA- dendrimers (commercialized as 3DNA-technology [119]) offer numerous possibilities for oligonucleotide detection, since the terminal polynucleotide strands can be varied and labeled with hundreds of radioactive or fluorescent labels. 

DNA-dendrimers have been used to identify oligonucleotides, for signal amplification in DNA microarray technology, for routine use in high-throughput functional genomic analysis, and as biosensors for the rapid diagnosis of genetic and pathogenetic diseases [120–123]. 

In a similar manner, radiolabeled monoclonal antibodies with high specific activity have been prepared by attachment of PAMAM dendrimers loaded with 111In or 153Gd complexes [124].

In vivo oxygen imaging is a strategy that offers the potential for diagnosing complications from diabetes and peripheral vascular diseases, as well as the detection of tumors and the design of their therapeutic treatment. 

The technique is based on the quenching of phosphorescence by oxygen and requires the presence of a chromophor (i.e., palladium-complexes of tetrabenzoporphyrins), which has strong absorption bands in the near-infrared range (620–900 nm wavelengths) to minimize the interference of natural chromophors present in the blood stream. The Pd complex has to be water-soluble and protected from interactions with serum-borne macromolecules such as albumin. 
It has been shown that encapsulation of Pd complexes into dendrimers of various sizes can be utilized to tune the  oxygen quenching of the phosphorescence. Although the dendritic shell used in these experiments appears to be too permeable to oxygen for optimized imaging purposes, there is some optimism in the viability of these dendrimer–Pd complexes in that the quenching constant has been influenced by a factor 5 [125].
Novel water-soluble dendritic nanoparticles, i.e., generation 4 PAMAM dendrimers with 3-N-[(NV,NVdimethylaminoacetyl) amino]-a-ethyl-2,4,6,-triiodobenzenepropanoic acid moieties covalently attached to the surface (G-4-(DMAA-IPA)37), have been applied in computed tomography (CT) imaging. The high iodine content (33%) makes them superior agents compared to iodinated small molecules for this technology,which provides a reliable and widely available imaging method with high spatial resolution.

Furthermore, the longer circulatory retention times of these macromolecular agents allow for an extended imaging time scale and potential reduced toxicity that would accompany repeated injections of high doses of small iodine molecules [126]. 

In summary, dendrimers and dendrons have an immense potential in the area of molecular imaging with clear leads and many opportunities yet to be explored.

8. Dendrimers as nano-scaffolds
The surface of dendrimers provides an excellent platform for the attachment of cell-specific ligands, solubility modifiers, stealth molecules reducing the interaction with macromolecules from the body defense system, and imaging tags (Fig. 6). The ability to attach any or all of these molecules in a welldefined and controllable manner onto a robust dendritic surface clearly differentiates dendrimers from other carriers such as micelles, liposomes, emulsion droplets, and engineered particles. 

One example of cell-specific dendritic carriers is a dendrimer modified with folic acid. The membraneassociated folate receptor is a folate binding protein that is over-expressed on the surface of a variety of cancer cells; therefore, folate modified dendrimers would be expected to internalize into these cells preferentially over normal cells via receptor-mediated endocytosis.

Folate–dendrimer conjugates have been shown to be well-suited for targeted, cancer-specific drug delivery of cytotoxic substances [127–129]. 

Very recently, folate–PAMAM dendrimers have been successfully used as carriers of boron isotopes in boron neutron-capture treatment of cancer tumors [130]. In addition to folates, carbohydrates constitute another important class of biological recognition molecules, displaying a wide variety of spatial structures due to their branching possibility and anomericity. 
To achieve sufficiently high binding affinities between simple mono- and oligosaccharide ligands and cell membrane receptors, these ligands have to be presented to the receptors in a multivalent or cluster fashion [131,132]. 

The highly functionalized surface of dendrimers provides an excellent platform for such presentations. The design, synthesis, and biomedical use of glycol-dendrimers as well as their application in diagnostic and for vaccinations have been thoroughly reviewed recently [133–139].

For example, the Thomsen–Friedenreich carbohydrate antigen (T-antigen), h-Gal-(1-3)-a-GalNAc,which has been well documented as an important antigen for the detection and immunotherapy of carcinomas, especially relevant to breast cancer, has been attached to the surface of PAMAM and other dendrimers [140–142]. 

An enhanced binding affinity was observed for all glycodendrimers. These constructs could have potential in blocking the metastatic sites of invasion tumor cells. 

A series of dendritic h-cyclodextrin derivatives bearing multivalent mannosyl ligands has been prepared and their binding efficiency towards the plant lectin concanavalin A (Con A) and a mammalian mannose-specific cell surface receptor from macrophages has been studied. 

The effects of glycodendritic architecture on binding efficiency, molecular inclusion, lectin-binding properties, and the consequence of complex formation using the anticancer drug docetaxel on biological recognition were investigated [143]. 

Di- to tetravalent dendritic galabiosides, carrying (Gala1–4Gal) moieties on their surfaces, were studied as inhibitors of pathogens based on bacterial species such as E. coli and Streptococcus suis.

Attachment of dendritic galabiosides onto cell surfaces would be expected to inhibit the attachment of bacteria using the same sugar ligand–receptor interactions.

The study revealed a clear enhancement of the binding affinity between glycodendrons and cell surfaces with increasing number of sugar moieties [144]. 

In a similar approach, glycodendrons carrying two to four h-d-galactose moieties on their surface, while the dendron core was connected to a proteindegrading enzyme, were synthesized. These glycodendriproteins are expected to attach to the surface of bacteria, allowing the enzyme to degrade the bacterial adhesin hence rendering the bacteria incapable of attaching to cell surfaces [145]. 

Anionic PAMAM dendrimers (G=3.5) were conjugated to d(+)-glucosamine and d(+)-glucoseamine 6-sulfate. These water-soluble conjugates not only revealed immunomodulatory and antiangiogenic properties but synergistically prevented scar tissue formation after glaucoma filtration surgery. In a validated and clinically relevant rabbit study, the long-term success rate was increased from 30% to 80% using these dendrimer conjugates [146].

The surfaces of PAMAM dendrimers, generations 0 to 3, were decorated with benzylpenicillin in an attempt to develop a new in vitro test to quantify IgE antibodies to specific h-lactam conjugates with the goal of improving the existing methods for diagnosing allergy to this type of antibiotic.  The monodispersity of dendrimers is advantageous over conventional peptide carrier conjugates such as human serum albumin (non-precise density of haptens in their structure) and poly-l-lysine (mixture of heterogeneous molecular weight peptides). Preliminary radioallergosorbent tests (RAST), using sera from patients allergic to penicillin, have confirmed the use of ulness of penicilloylated dendrimers [147].

Monolayers formed by generation 4 PAMAM dendrimers on a gold surface were functionalized with biotin and produced a biomolecular interface that was capable of binding high levels of avidin. Avidin binding as high as 88% coverage of the surface was observed despite conditions that should cause serious steric hindrance. These dendritic monolayers were utilized as a model to study protein–ligand interactions [148]. 

Another approach utilizing dendrimers as nano-scaffolds involved functionalization of the surface of poly(propylene imine) dendrimers by attachment of adamantane-bis(propylurea) and palmitylbis( propylurea) groups, as well as their respectivethiourea derivatives. These dendrimers were able to bind glycine–urea containing guest molecules to their surface based on secondary hydrogen bonds, using the propyl(thio)urea surface groups as pincers. The thiourea derivatives formed somewhat stronger bonds with the guest molecules. Furthermore, dendrimers of higher generation, carrying a higher density of surfacegroups, formed more stable dendrimer-guest constructs [40].

Fig. 6. Schematic presentation of dendrimers as nano-scaffold for the attachment of cell-specific ligands, modifiers, and fluorescence tags.

9. Dendrimers as nano-drugs
Dendrimers have been studied extensively as antitumor, antiviral, and antibacterial, drugs [56]. As antitumor drugs, dendrimers have mainly been used in photodynamic therapy (PDT). In one approach, they are constructed around a light harvesting core (i.e., a porphyrin) [149]. 

To reduce the toxicity under nonirradiative conditions (dark toxicity), these dendrimers are encapsulated into poly(ethylene glycol)-b-poly (aspartic acid) micelles. These micelles are stable under physiological conditions at 6.2 to 7.4, however, disintegrate in the acidic environment (pH ~5.0) of the intracellular endosomal compartment [150]. 

Alternatively, the photosensitizer 5-aminolevulinic acid has been attached to the surface of dendrimers and studied as an agent for PDT of tumorigenic keratinocytes [151]. 

Photosensitive dyes have been incorporated into dendrimers and utilized in PDT devices. For example, uptake, toxicity, and mechanism of photosensitization of the dye pheophorbide a (pheo) was compared to its complex with diaminobutane poly (propylene imine) (DAB) dendrimers in human leukemia cells in vitro [152].

Poly(lysine) dendrimers modified with sulfonated naphthyl groups have been found to be useful as antiviral drugs against the herpes simplex virus [153]. Such a conjugate based on dendritic poly(lysine) scaffolding is VivaGelk, a topical agent currently under development by Starpharma Ltd., Melbourne, Australia, that can potentially prevent/reduce transmission of HIV and other sexually transmitted diseases (STDs). VivaGelk (SPL 7013) is being offered as a water-based gel, with the purpose to prevent HIV from binding to cells in the body. 

The gel differs from physical barriers to STDs such as condoms by exhibiting inhibitory activity against HIV and other STDs. In July 2003, following submission of an Investigational New Drug (IND) application, Starpharma gained clearance under U.S. FDA regulations to proceed with a Phase I clinical study to assess the safety of VivaGelk in healthy human subjects. 

This Phase I study, representing the first time a dendrimer pharmaceutical has been tested in humans, compared 36 women who received either various intra-vaginal doses of VivaGelk or a placebo gel daily for one week. The trial was double blinded so that the volunteers, principal investigator and Starpharma did not know who was receiving placebo or VivaGelk. Study participants were assessed for possible irritant effects of the gel. 

Additionally, the women were assessed for any possible effect upon vaginal microflora (natural micro-organisms in the vagina) or absorption into the blood of the active ingredient of VivaGelk. A thorough review of the complete data revealed no evidence of irritation or inflammation. Preclinical development studies had demonstrated that VivaGelk was 100% effective at preventing infection of primates exposed to a humanized strain of simian immunodeficiency virus (SHIV) [154]. 

In earlier studies, it was found that PAMAM dendrimers covalently modified with naphthyl sulfonate residues on the surface, also exhibited antiviral activity against HIV. This dendrimer-based nano-drug inhibited early stage virus/cell adsorption and later stage viral replication by interfering with reverse transcriptase and/or integrase enzyme activities [155,156].

The general mode of action of antibacterial dendrimers is to adhere to and damage the anionic bacterial membrane, causing bacterial lysis [56,157]. PPI dendrimers with tertiary alkyl ammonium groups attached to the surface have been shown to be potent antibacterial biocides against Gram positive and Gram negative bacteria. The nature of the counter ion is important, as tetraalkyl- ammonium bromides were found to be more potent antibacterials over the corresponding chlorides [158]. 

Poly(lysine) dendrimers with mannosyl surface groups are effective inhibitors of the adhesion of E. coli to horse blood cells in a haemagglutination assay, making these structures promising antibacterial agents [159]. 

Chitosan– dendrimer hybrids have been found to be useful as antibacterial agents, carriers in drug delivery systems, and in other biomedical applications. Their behavior has been reviewed very recently [160]. Triazine-based antibiotics were loaded into dendrimer beads at high yields. The release of the antibiotic compounds from a single bead was sufficient to give a clear inhibition effect [161]. In many cases, dendritic constructs were more potent than analogous systems based on hyperbranched polymers.

10. Biocompatibility of dendrimers
Dendrimers have to exhibit low toxicity and be non-immunogenic in order to be widely used in biomedical applications. To date, the cytotoxicity of dendrimers has been primarily studied in vitro, however, a few in vivo studies have been published [56]. 

As observed for other cationic macromolecules, including liposomes and micelles, dendrimers with positively charged surface groups are prone to destabilize cell membranes and cause cell lysis. For example, in vitro cytotoxicity IC50 measurements (i.e., the concentration where 50% of cell lysis is observed) for amino-terminated PAMAM dendrimers revealed significant cytotoxicity on human intestinal adenocarcinoma Caco-2 cells [162,163]. 

Furthermore, the cytotoxicity was found to be generation-dependent, with higher generation dendrimers being the most toxic [162,164]. 

A similar generation dependence of amino-terminated PAMAM dendrimers was observed for the haemolytic effect, studied on a solution of rat blood cells [165]. However, some recent studies have shown that amino-terminated PAMAM dendrimers exhibit lower toxicity than more flexible aminofunctionalized linear polymers perhaps due to lower adherence of the rigid globular dendrimers to cellular surfaces. The degree of substitution as well as the type of amine functionality is important, with primary amines being more toxic than secondary or tertiary amines [164]. 

Amino-terminated PPI and PAMAM dendrimers behave similarly with regard to cytotoxicity and haemolytic effects, including the generation dependent increase of both [70,165].

Comparative toxicity studies on anionic (carboxylate- terminated) and cationic (amino-terminated) PAMAM dendrimers using Caco-2 cells have shown a significantly lower cytotoxicity of the anionic compounds [162]. In fact, lower generation PAMAM dendrimers possessing carboxylate surface groups show neither haematotoxicity nor cytotoxicity at concentrations up to 2 mg/ml [165]. 

The biocompatibility of dendrimers is not solely determined by the surface groups. Dendrimers containing an aromatic polyether core and anionic carboxylate surface groups have shown to be haemolytic on a solution of rat blood cells after 24 h. It is suggested that the aromatic interior of the dendrimer may cause haemolysis through hydrophobic membrane contact [165].

One way to reduce the cytotoxicity of cationic dendrimers may reside in partial surface derivatization with chemically inert functionalities such as PEG or fatty acids. The cytotoxicity towards Caco-2 cells can be reduced significantly (from IC50 ~0.13 mM to N1 mM) after such a modification. This observation can be explained by the reduced overall positive charge of these surface-modified dendrimers. A partial derivatization with as few as six lipid chains or four PEG chains on a G4-PAMAM, respectively, was sufficient to lower the cytotoxicity substantially [163]. 

In studies conducted at DNT using Caco-2 and two other cell lines it was found that, besides (partial) PEGylation of the surface, surface modification with pyrrolidone, another biocompatible compound, can significantly reduce cytotoxicity to levels far better than those of currently available products [166]. In some cases, the cytotoxicity of PAMAM dendrimers could be reduced by additives such as fetal calf serum [74].

Only a few systematic studies on the in vivotoxicity of dendrimers have been reported so far. Upon injection into mice, doses of 10 mg/kg of PAMAM dendrimers (up to G=5), displaying either unmodified or modified amino-terminated surfaces, did not appear to be toxic [153,167]. Hydroxy- or methoxy-terminated dendrimers based on a polyester dendrimer scaffold have been shown to be of low toxicity both in vitro and in vivo. 

At very high concentrations (40 mg/ml), these polyester dendrimers induced some inhibition of cell growth in vitro but no increase in cell death was observed. Upon 

4. Future Outlook
One of the most promising aspects of the work described here involves the synthesis and characterization of structured bimetallic nanoparticles (Scheme 4). In the future we hope to show that it is possible to synthesize particular structures based on a first-principles understanding of catalytic selectivity. 

This goal will require, among other things, better synthetic methodologies for nanoparticle fabrication. Some guidance as to how this might be accomplished comes from Yamamoto and coworkers who have studied the interaction of SnCl2, as well as other metal salts, with phenylazomethine dendrimers.92 They found that SnCl2 complexes with the imine groups of the dendrimer in a stepwise radial fashion.92 Such precise spatial control of metal loading could potentially be used to synthesize multishell nanoparticles for catalytic and other applications.93 Another interesting avenue for further research is the exploration of DEN catalysts as natural enzyme mimics; that is, systems in which several catalytic sites can act in concert to yield desirable products. 

Indeed, we previously found that when Cu DENs are converted to Ag DENs by redox displacement (eq 1), Cu2+ remains complexed within the dendrimer over a broad range of pHs.60 This suggests that dendrimer-based catalysts could be designed for reactions that require the cooperative action of a zerovalent metal catalyst and an ionic co-catalyst in close proximity. A well-known example of such a reaction, which we are presently studying,94 is the Stille cross-coupling reaction between aryl and vinyl halides with organo stannanes using a zerovalent Pd catalyst. It has been shown that Cu(I) salts have a significant co-catalytic effect in this reaction.95 Since its origins in solid-phase peptide synthesis, combinatorial chemistry has impacted fields ranging from catalysis to agriculture as a route to more cost-effective product development. 96 

In its simplest form, combinatorial chemistry involves preparation of a large number of related chemical entities from a relatively small number of building blocks, and then subsequent screening of these entities for activity. Building DENs through combinatorial chemistry might be an attractive route to synthesize multi-metallic alloys having well-defined particle sizes and tunable solubility as improved catalysts for such reactions as the electrooxidation of methanol in fuel cells or ester hydrolysis. 

DEN libraries consisting of metal catalysts and ionic co-catalysts could also be prepared. Such a combinatorial study has already been reported for constructing a combinatorial library of polymer-bound catalyst contenders.97 In addition to catalysis, there are other fields that might benefit from DENs. For example, dendrimer-encapsulated semiconductor nanoparticles (or quantum dots, QDs) having controlled sizes, and thus well-defined luminescence properties, have already been prepared.61 

Such materials might be useful as biological labels, because QDs have highly desirable characteristics compared to organic dyes98 and because the luminescent properties of QDs are not affected by the surrounding dendrimers to an appreciable extent. Moreover, the dendrimer itself possesses many functional groups on its periphery that can be used as handles for attachment to biomolecules or surfaces. 
Additional possibilities for using DENs include the integration of nanoparticles into dendrimers that can harvest light and transfer the energy to a reaction center.99 Such composite DENs could be used for light-induced hydrogen evolution using colloidal Pt or bimetallic catalysts.100,101 Another possible application is the formation of magnetic DENs, composed of metals or bimetallic alloys,102 that could find use in the field of data storage.

Figure 8. HRTEM micrographs and size-distribution histograms for (a),(b) G4-OH(Pd40) and (c),(d) Pd40 MPCs (MPC-Pd40) after extraction with a toluene solution containing 500 íM n-hexanethiol. Reprinted with permission from J. Am. Chem. Soc. 2003, 125, 11190-11191. 

Figure 9. Photographs illustrating the selective extraction of DENs. The vial on the left contained a mixture of 0.20 mM G4-OH(Au147) and 0.18 mM G4-OH(Ag110) DENs in water. When an n-hexane solution containing 0.25 M n-decanoic acid was added and the vial shaken, the Ag nanoparticles were extracted into the organic phase as monolayerprotected clusters. Reprinted with permission from Chem. Mater. 2004, 16, 4202-4204. Copyright 2004 American Chemical Society. 702 J. Phys. Chem. B, Vol. 109, No. 2, 2005 Scott et al.

5. Summary and Conclusions
This article has provided an overview of progress in the synthesis and characterization of dendrimer-encapsulated nanoparticles. We have shown that these materials can serve as models for addressing scientific questions and that they may also have some technological significance. It is worthwhile to summarize what we know and what we do not know about DENs. We know, for example, that metal ions having an affinity for tertiary amines are sequestered within the interior of PAMAM and PPI dendrimers. 

Following chemical reduction, HRTEM, SAXS, and catalysis experiments confirm that these ions are reduced to atoms and that the atoms aggregate into nanoparticles having sizes that reflect the original metal-ion-to-dendrimer ratio. TEM indicates that DENs can be quite monodisperse in size, but there are some limitations of this method that make it necessary to qualify this statement. First, TEM does not clearly resolve particles smaller than _1 nm, which means that if particles in this size range are present they are not counted in particle-size distributions. 

Likewise, the inherent resolving power of the TEM used to obtain the data reported here is (0.19 nm, and therefore it is difficult to draw definitive conclusions regarding monodispersities of DENs that approach this value. Clearly, better methods are required for measuring particle sizes (and shapes) in the range of 0-3 nm. In addition to monometallic DENs, it is also possible to prepare bimetallic materials that have either random alloy or core/shell structures. DENs are stable for long periods of time, although some metals, such as Cu and Ag, easily oxidize in air. 

This is partly a consequence of the fact that the redox potential of metals shift positive as their size decreases.103 DENs can be rendered soluble in water, organic and fluorous solvents, and even in liquid and supercritical CO2, and this provides a major opportunity to study metal-particle-catalyzed reactions in unusual solvent environments. We also know that DENs can be extracted from within the interior of dendrimers without aggregation and without major changes in size or chemical composition. Selective extraction experiments also provide a sensitive means for studying the structure of bimetallic core/ shell DENs. With regard to catalysis, we know that dendrimers can act as size-selective “nanofilters” that confer a degree of substrate selectivity to intrinsically nonselective metal catalysts. 

However, as with all selective filters there is a cost, and in this case the cost is reduced turnover frequency. This means that the rates of reactions catalyzed by DENs are often rather slow. We know that DENs catalyze hydrogenation and carbon-coupling reactions, but the full scope of reactions that can be catalyzed by DENs is unknown. For example, it seems reasonable to expect that dendrimers could be prepared that exhibit regioselectivity and enantioselectivity. 

We know that catalytic reactions occurring within DENs can lead to unusual product distributions, but the scope and underlying reasons for this observation are not known. We also know that catalytically active DENs can be recycled by attaching appropriate functional groups on the periphery of the dendrimer. We know that DENs can be immobilized on surfaces and used for electrocatalysis and other forms of heterogeneous catalysis. 

We also know that in the absence of solvent or in the presence of a poor solvent, dendrimers collapse around the encapsulated metal nanoparticle and that this shuts off catalytic activity. However, surface-confined DENs can be activated by removing the dendrimer, and in favorable cases this does not lead to too much aggregation. There are a few other interesting questions about DENs that have not yet been addressed. For example, can DENs provide a platform for studying catalytic quantum-size effects in homogeneous solution or as supported catalysts?104,105 Is it possible to control the shape as well as the size of nanoparticles using dendrimer templates? What does the surface of a DEN look like? That is, what percentage of the atoms on the surface of a DEN are catalytically active? Although we have some idea where DENs are located inside dendrimers, it would be interesting to know the distribution of locations and how the presence of the nanoparticle affects the dynamics of dendrimer motion. Clearly, there are many interesting questions about DENs that remain unanswered. We hope this article will stimulate others to bring their particular expertise to bear on DENs and related materials. It would be particularly interesting to examine the properties of nanoparticles encapsulated in other families of dendrimers, and it would also be useful to devise characterization methods that would provide additional insight into the properties of DENs.

11. Conclusions
The high level of control over the architecture of dendrimers, their size, shape, branching length and density, and their surface functionality, makes these compounds ideal carriers in biomedical applications such as drug delivery, gene transfection and imaging. The bioactive agents may either be encapsulated into the interior of the dendrimers or they may be chemically attached or physically adsorbed onto the dendrimer surface, with the option to tailor the properties of the carrier to the specific needs of the active material and its therapeutic applications.

Furthermore, the high density of surface groups allows attachment of targeting groups as well as groups that modify the solution behavior or toxicity of dendrimers. Surface modified dendrimers themselves may act as nano-drugs against tumors, bacteria, and viruses. Recent successes in simplifying the synthesis of dendrimers such as the dlegoT and dclickT approaches have provided a vastly expanded variety of dendritic compounds while at the same time reducing the cost of their production. This review of biomedical applications of dendrimers clearly illustrates the potential of this new bfourth architectural class of polymersQ and substantiates the high optimism for the future of dendrimers in this important field.

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