1-Origin of Dendrimers:

Polymer chemistry and technology have traditionally focused on linear polymers,

which are widely in use. Linear macromolecules only occasionally contain some
smaller or longer branches. In the recent past it has been found that the properties of
highly branched macromolecules can be very different from conventional polymers.
The structure of these materials has also a great impact on their applications. First
discovered in the early 1980’s by Donald Tomalia and co-workers, these hyper
branched molecules were called dendrimers. The term originates from ‘dendron’
meaning a tree in Greek. At the same time, Newkome’s group independently reported
synthesis of similar macromolecules. They called them arborols from the Latin word
‘arbor’ also meaning a tree. The term cascade molecule is also used, but ‘dendrimer’
is the best established one.
Dendrimer chemistry began nearly two decades ago with the synthesis and
characterization of a family of highly branched macromolecules.( Gorman and Smith)( Gorman
et al)The general Dendrimer structure consisted of an inner core molecule with hyper
branched polymeric structures, known as dendrons, extending outward from the core.
Each dendritic branch was terminated with a chosen periphery structure , (Fig. 1) The
ability to synthesize well-defined monodisperse macromolecules using repetitive
activate/couple growth cycles added a new dimension to polymer chemistry.
At the nanoscale (molecular level),there are relatively few natural examples of this
architecture. Most notable are glycogen and amylopectin, macromolecular hyper
branched structures that nature uses for energy storage.( Donald A. Tomalia)
The complexity of organic synthesis has been steadily enhanced by utilizing the
known hybridization states of carbon and specific heteroatom to produce key
molecular-level hydrocarbon building blocks (modules) and functional groups
(connectors),These two construction parameters have been used to assemble literally
millions of more complex structures, Relatively small (i.e. !1 nm) molecules were
produced, the structures of which could be controlled as a function of their shape,
mass, flexibility, and functional group placement, Based on the various hybridization
states of carbon, at least four major carboskeletal architectures are known (Corey,
Cheng ) (Heilbronner, Dunitz) They are recognized as the
(I) linear, (II) bridged (2D/3D), (III) branched and , more recently, the (IV) dendritic
(cascade) (Buhleier et al) (Fig.2).
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 (cascade) subclasses: (a) random
hyper branched polymers, (b) dendrigraft polymers, (c) dendrons, and (d) Dendrimers
(Fig. 3).
The order of this subset, from (a) to (d), reflects the relative degree of structural
control present in each of these dendritic architectures ( Tomalia et al).All dendritic
polymers are open, covalent assemblies of branch cells (Fig. 3a). They may be
organized as very symmetrical, monodispersed arrays, as is the case for Dendrimers,
or as irregular, polydispersed assemblies that typically define random, hyper branched
polymers.
The dendritic structure is characterized by ‘layers’ between each focal point (or
cascade) called generations (shown as circles on Fig.4) ( Supattapone et al). The exact
numbering of generations has been the subject of some confusion , the dendrimer
generation is defined as the number of focal points (cascade points) when going from

1
the core to the surface, a generation 5 (G5) dendrimer thus has 5 cascade points
between the core and the surface.
The core is sometimes denoted generation ‘zero’ (G0), as no cascade points are
present, for a polypropylene imine (PPI) dendrimer, the core is 1,4-diaminobutane
which has no cascade points, for a polyamido amine (PAMAM) ‘Starburst™’
dendrimer the core is ammonia etc. (hydrogen substituents are not considered
a focal point). In PAMAM dendrimers the intermediate compounds having
carboxylate surface groups are denoted half-generation dendrimers, that is dendrimers
of e.g. G1.5 or G2.5.
The dendrimer design can be based on a large variety of linkages, such as polyamines
(PPI dendrimers), a mix of polyamides and amines (PAMAM dendrimers) or built up
by more hydrophobic poly(aryl ether) subunits (C. J. Hawker et al).
More recent examples are dendrimer designs based on carbohydrate or calixarene core
structures, or containing ‘third period’ elements like silicon or phosphorus (J. P. Majoral and
A. M. Caminade), just to give a few examples (Fig.5).

2-Dendrimer synthesis:
In contrast to traditional polymers, dendrimers are unique core–shell structures
possessing three basic architectural components : (I) a core, (II) an interior of shells
(generations) consisting of repeating branch-cell units, and (III) terminal functional
groups (the outer shell or periphery) ( Donald A. Tomalia).

2.1-"Divergent" Dendrimer Growth:

The development of new synthetic schemes enabled manipulation of the structure of

dendrimers. One scheme, the divergent strategy, In the divergent methods, dendrimer
grows outwards from a multifunctional core molecule.
The core molecule reacts with monomer molecules containing one reactive and two
dormant groups giving the first generation dendrimer. Then the new periphery of the
molecule is activated for reactions with more monomers. The process is repeated for
several generations and a dendrimer is built layer after layer (Fig.6). The divergent
approach is successful for the production of large quantities of dendrimers. Problems
occur from side reactions and incomplete reactions of the end groups that lead to
structure defects. To prevent side reactions and to force reactions to completion large
excess of reagents is required. It causes some difficulties in the purification of the
final product.

2.2-"Convergent" Dendrimer Growth:

To eliminate this problem, a convergent strategy for the synthesis of dendrimers was
developed. This strategy originates with the peripheral units and uses the same
iterative deprotection and coupling scheme to construct the dendritic branches inward
(Fig.7).
Each coupling cycle designates a new layer of branch points referred to as a
generation. The dendritic arms are ultimately terminated with a linking unit that is
used for core attachment. The convergent growth method has several advantages. It
is relatively easy to purify the desired product and the occurrence of defects in the
final structure is minimized.

2
Monodispersity becomes a problem at high molecular weights therefore, the
convergent strategy is not preferred when synthesizing large generation dendrimers.

2.3-"Hypercores" and "Branched Monomers":

In the early 1990s, the area of synthetic methodology in dendrimer research was led
by the Fréchet group at Cornell University. After the development of the convergent
approach, their efforts focused on the acceleration of dendrimer syntheses. The
outcome of this research was the demonstration (Fig. 8) of 'hypercores' and 'branched
monomers'. These methods involve the pre-assembly of oligomeric species which can
then be linked together to give dendrimers in fewer steps or higher yields. Hypercores
and branched monomers allow the chemist to devise synthetic strategies that are more
convergent in the classical synthetic sense of the word. ( AN Shipway)

2.4-"Double Exponential" and "Mixed" Growth:

The most recent fundamental breakthrough in the practice of dendrimer synthesis has
come with the concept and implications of 'double exponential' growth (Fig. 9).
Double exponential growth, similar to a rapid growth technique for linear polymers,
involves an AB2 monomer with orthogonal protecting groups for the A and B
functionalities. This approach allows the preparation of monomers for both
convergent and divergent growth from a single starting material. These two products
are reacted together to give an orthogonally protected trimer, which may be used to
repeat the growth process again. The strength of double exponential growth is more
subtle than the ability to build large dendrimers in relatively few steps. In fact, double
exponential growth is so fast that it can be repeated only two or perhaps three times
before further growth becomes impossible. The double exponential methodology
provides a means whereby a dendritic fragment can be extended in either the
convergent or the divergent direction as required. In this way, the positive aspects of
both approaches can be accessed without the necessity to bow to their shortcomings.
( AN Shipway)

3-MOLECULAR STRUCTURE:
The dendrimer structure can be divided into three parts:
–The multivalent surface, with a high number of potential
reactive sites.
–The ‘outer shell’ just beneath the surface having a well-defined
microenvironment protected from the outside by the dendrimer
surface.
–The core, which in higher generation dendrimers is protected
from the surroundings, creating a microenvironment surrounded by the dendritic
branches (C. J. Hawker et al).
These three architectural components (core, interior, and periphery) 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, whereas the terminal functional
groups increase exponentially as a function of generation.

3
As a consequence, lower generations are generally open, floppy structures, whereas
higher generations become robust, less deformable spheroids, ellipsoids, or cylinders
depending on the shape and directionality of the core (Donald A. Tomalia) (Fig.10).
These structures are governed by many factors including solvation, sterics, hydrogen
bonding, stereo centers, and benzyl substitution patterns ( Scherrenberg et al)( Gorman and Smith).
The interior is thus well-suited for encapsulation of guest molecules. The three parts
of the dendrimer can be tailored specifically for the desired purposes, e.g. as dendritic
sensors, drug vehicles or drugs. The multivalent surfaces on a higher-generation
dendrimer can contain a very high number of functional groups. This makes the
dendritic surfaces and outer shell well-suited to host–guest interactions where the
close proximity of a large number of species is important.
As the chains growing from the core molecule become longer and more branched (in
4 and higher generations) dendrimers adopt a globular structure (Caminati et al) .
Dendrimers become densely packed as they extend out to the periphery, which
forms a closed membrane-like structure. When a critical branched state is reached
dendrimers cannot grow because of a lack of space. This is called the ‘starburst
effect’(Fischer and Vögtle). The increasing branch density with generation is also believed
to have striking effects on the structure of dendrimers. They are characterized by the
presence of internal cavities and by a large number of reactive end groups (Fig.11).

4-Properties of dendrimers:
4.1-Characterization:

4.1.1-Size and size distribution:

The molar mass of the dendrimer can be predicted

mathematically (Tomalia et al) :

where: Mc : is the molar mass of the core, Mm : the molar mass of the branched
monomer, Mt : the molar mass of the terminal groups, nc : the core multiplicity, nm :
the branch-juncture multiplicity, G : the generation number.
The increase of the number of dendrimer terminal groups is consistent with the
geometric progression:

The monodispersed nature of dendrimers, has been verified

extensively by mass spectrometry, size-exclusion chromatography,
gel electrophoresis, and electron microscopy (TEM) (Jackson et al). as
illustrated by TEMs for a Gen 5–10 series of PAMAM dendrimers (Fig. 12).

4
Additional techniques to further elucidate structure-property relationships of
dendrimers include fluorescence quenching, luminescence, and electrochemistry.
Dendrimers pertain both to the molecular chemistry world for their step by step
controlled syntheses, and to the polymer world because of their repetitive structure
made of monomers; thus they benefit from analytical techniques from both worlds.

4.1.2-Structural Elucidation:

The ultimate proof of molecular structure is often reached by the determination of a

crystal structure, In the case of dendrimers, however, this technique is of little use.
The polymeric nature of dendrimers generally leaves their solid-state structure without
any long-range order. Even when some degree of structure is present for instance in
the case of very dense-surfaced spherical dendrimers there is such a degree of disorder
within the inner generations that a crystal structure cannot be determined. There are a
few examples of crystalline dendrimers and powder and single crystal.
" X-ray studies of dendrimers", but these are confined to low generations and rigid,
hindered molecules.
Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry have
both been invaluable techniques in the characterization of dendrimers. Indeed, 1H and
13 C NMR spectra of dendrimers can be surprisingly simple and contain a great deal
of information about defects and impurities in their structures.
The mass spectrometry of dendrimers has benefited from the soft ionization
techniques developed for the study of large biomolecules.
Other methods, familiar to polymer chemists, have been used extensively in the
characterization of dendrimers. Electron microscopy has been of great use in the
visualization of dendrimers, and their aggregates, and Gel Permeation
Chromatography (GPC) has been used for the calculation of radii of gyration,
hydrodynamic radii, and polydispersities. Low Angle Laser Light Scattering
(LALLS), Small Angle Neutron (SANS), and X-ray (SAXS) Scattering techniques
have met with limited use.( AN Shipway)

4.1.3-Characterisation of the Dendritic Microenvironment:

Many investigators have made use of functional probes in order to study dendritic
microenvironments. These probes can either be attached covalently to the dendritic
structure, like the photochemical, chiral, and solvatochromic moieties , or they can be
introduced as guest species.
Spectroscopic methods have also produced information about dendritic
microenvironments. PAMAM dendrimers have been shown20 to have decreased 13 C
relaxation times for internal generations, suggesting that these moieties are less
mobile than the surface groups.
Rotational-Echo Double Resonance (REDOR), solid-state NMR spectroscopy has
been used to examine the shape of the Fréchet polyethers, and Electron Spin
Resonance (ESR) spectroscopy of complexed PAMAMs have been examined.
Computer modelling of dendrimers has been used extensively for the purposes of
visualization and dynamics experiments.( AN Shipway)
This is review of the main analytical techniques used for the characterization of the
chemical composition, the morphology, the shape, and the homogeneity of
dendrimers. It includes NMR, IR, Raman, UV–Visible, fluorescence, circular

5
dichroism, X-ray diffraction, mass spectrometry, SAXS, SANS, Laser Light
Scattering, microscopy, SEC, EPR, electrochemistry, electrophoresis, intrinsic
viscosity, DSC, and dielectric spectroscopy.

4.1.4-Analytical methods:

A-Size Exclusion Chromatography (SEC):

Analysis was performed using an Alliance Waters 2690 separation module equipped
with a Waters UV-Vis detector, a Wyatt Dawn laser photometer, Optilab
interferometric refractometer and Waters ultrahydrogel columns. Phosphate buffer
(0.05 M, pH 2.5) with 0.025% sodium azide was used as mobile phase. The flow rate
was maintained at 0.6 mL/min. Sample concentration was kept at 2 mg/mL and 100
μL was injected. Data was elaborated using Astra and PeakFit software.

B-Potentiometry:

Dendrimers were dissolved in NaCl (0.1 M) solution at concentration of 0.5 mg/mL,

pH was set to around 3. In case of each sample the same number of moles (adjusted
by the volume) was titrated. Titrations were performed at room temperature, under
nitrogen atmosphere, using Molspin automated titration system, Mettler-Toledo
combination In Lab electrode and NaOH (0.1 M) as a titrant.

C-Gel electrophoresis:

Dendrimers was performed using a vertical electrophoresis system (Model FB-VE10-

1 Fisher Biotech) with a commercial power supply (Model EC135-90; Thermo
Electron Corporation).

D-Matrix-assisted laser desorption ionization-time of flight mass

spectrometry:

Spectra of dendrimer nanodevices were recorded on a Bruker Biflex IV

spectrophotometer, using 2,5-dihydroxybenzoic acid (DHB) as a matrix. First 0. 5 μL
of matrix was placed on target plated, and evaporated. Then 0.5 μL of the dendrimer
(~1.5 mg/mL) was spotted over the matrix. Matrix and dendrimer were dissolved in
75% ACN and 0.1% TFA aqueous solution. For each spectrum 100 shots and 50% of
laser power was applied.

E-Nuclear Magnetic Resonance:

Spectra were recorded using Bruker AMX 400 MHz, D2O as solvent and at sample
concentrations 20 mg/mL.( Wojciech et al).

4.2-Physical and chemical properties:

Dendrimers are monodisperse macromolecules, unlike linear polymers. The classical

polymerization process which results in linear polymers is usually random in nature
and produces molecules of different sizes, whereas size and molecular mass of
dendrimers can be specifically controlled during synthesis.

6
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 due
to failed couplings (Fre´chet et al). Because of their molecular architecture, dendrimers
show some significantly improved physical and chemical properties when compared
to traditional linear polymers.
Dendrimers are of interest for their unusual physical properties. Much work has been
carried out in areas such as melt viscosity, glass transition temperature, rheological
properties and even on the photo induced electron transfer to C60.
The most exciting physical property of dendrimers is the variation of their intrinsic
viscosities with molecular weight. It is found that, when the generation increases
beyond a certain point, the intrinsic viscosity begins to decline, contrary to the
behavior of linear polymers (Curve. 1). This effect is believed to be a consequence of
the globular shapes of high generation dendrimers leaving them unable to 'tangle' with
one another after the manner of linear polymers. ( AN Shipway)
In solution, linear chains exist as flexible coils, in contrast, dendrimers form a
tightly packed ball. This has a great impact on their rheological properties. Dendrimer
solutions have significantly lower viscosity than linear polymers (Fréchet). When the
molecular mass of dendrimers increases, their intrinsic viscosity goes through a
maximum at the fourth generation and then begins to decline (Mourey et al). Such
behaviour is unlike that of linear polymers. For classical polymers the intrinsic
viscosity increases continuously with molecular mass.
The presence of many chain-ends is responsible for high solubility and miscibility and
for high reactivity (Fréchet). Dendrimers’ solubility is strongly influenced by the nature
of surface groups.
Dendrimers terminated in hydrophilic groups are soluble in polar solvents, while
dendrimers having hydrophobic end groups are soluble in nonpolar solvents.
Dendrimers have some unique properties because of their globular shape and the
presence of internal cavities. The most important one is the possibility to encapsulate
guest molecules in the macromolecule interior.
The observation of photo physical changes were the first evidence of encapsulation
behavior of dendrimers(Hawker et al)( Fréchet et al). Hawker et al. coupled Frechet-type
dendrons to a 4-(N-methylamino)-1- nitrobenzene core and observed an increase in
the solvatochromic shift in the absorption spectra as the dendrimer generation
increased (Hawker et al). The results indicated the larger generation dendrimers effectively
shielded solvent and created an intrinsic microenvironment around the core.
Photochemical modifications of the dendritic surface cause encapsulation and release
of guest molecules. A fourth generation polypropylene imine dendrimer with 32 end
groups was terminated in azobenzene groups (Fig.13). The azobenzene groups
undergo a fully reversible photoisomerization reaction. The E isomer is switched to
the Z form by 313 nm light and can be converted back to the E form by irradiation
with 254 nm light or by heating. Such dendrimers can play the role of
photoswitchable hosts for eosin Y.
Dendrimers are attractive candidate molecules for studying electron transfer based
solely on the intrinsic ability of the dendrons (for a given molecular weight) to
effectively shield the core with organic material. The distance of electron travel and
the material surrounding the core governs electron transfer rate (Gorman et al) (Fig.14) .
This distance can be altered by 1) increasing the bulk material of the dendrons around
the redox-active core, and/or 2) alter the dendritic architecture to create a dynamic
conformational change of the dendrimer. Both strategies would result in an increase in

7
the effective encapsulation and, subsequently, the distance of electron transfer to the
redox active core.
Already early in the history of dendrimers it was suggested that the 3-dimensional
nanosized structure of the higher generation dendrimers would make this class of
synthetic molecules suitable as mimics of proteins (Farin and Avnir).
however, that in contrast to proteins which consist of folded, linear polypeptide
chains, the branched architecture of the dendrimer interior is to a large extent formed
by covalent bonds, resulting in a somewhat less flexible structure. In addition, the
dendrimer is on average less compact than a protein, i.e. interior is not packed as
efficiently as in typical proteins, and the dendrimer contains a substantially higher
number of surface functional groups than proteins of comparable molecular weight.
(Table 1).
Molecular dynamic studies carried out by several research groups on dendrimers show
that the dendrimers, similar to proteins, can adapt ‘native’ (e.g. more tight) or
‘denaturated’ (e.g. extended) conformations dependent on the polarity, ionic strength
and pH of the solvent.
Amino-terminated PPI and PAMAM dendrimers (that is dendrimers having primary
amines as surface groups) exhibit extended conformations upon lowering of pH
because electrostatic repulsion between the protonated tertiary amines in the interior
as well as between the primary amines at the dendrimer surface, forces the dendrimer
branches apart (I. Lee et al) (Fig.15). At pH > 9 back-folding occurs as a consequence of
hydrogen bonding between the interior protonated tertiary amines and the primary
surface amines, resulting in a denser core (W. Chen et al).The pH-related conformational
changes are dependent on the nature of the charged group at the dendrimer surface.
For PPI dendrimers having surface carboxylic groups ‘small angle neutron scattering’
(SANS) and NMR measurements of the diffusion coefficients in aqueous buffer, show
that these dendrimers have the most extended conformations at pH 4 and pH 11,
(Fig.15) This may be due to electrostatic repulsion between the protonated cationic
inner tertiary amines at low pH, and electrostatic repulsion between the negatively
charged deprotonated carboxylates at the dendrimer surface at high pH forcing the
dendritic branches apart.
At pH 6 the carboxy-terminated PPI dendrimer has no net charge, resulting in a tighter
conformation controlled by intramolecular hydrogen bonding (W. Chen et al). Molecular
density measurements at this pH show a homogeneous molecular density over the
whole dendrimer, indicating a substantial degree of back-folding i.e. hydrogen
bonding between terminal groups and groups in the core region (Fig.16).
The polarity of the solvent greatly influences the 3-dimensional structure of
dendrimers, Initial theoretical studies by de Gennes and Hervet on unmodified
PAMAM (Starburst™) dendrimers using a self consistent mean-field model,
concluded that in good solvents (that is solvents with a high ability to solvate the
dendritic structure), the dendrimers had the highest molecular density at the periphery,
leading to dense packing of the surface groups upon increasing generation (Gennes and
Hervet).
Recent NMR studies performed on PPI dendrimers indicate that an a polar solvent
such as benzene will favour polar intramolecular interactions (e.g. hydrogen bonding)
resulting in back-folding of the dendrimer arms into the dendrimer interior, whereas
the increased acidity of chloroform, will increase solvation of the dendrimeric
structure via hydrogen bond donation to the interior tertiary amines resulting in a
more extended conformation of the dendrimer (M. Chai et al). Both theoretical as well as
experimental studies on amino functionalized PPI and PAMAM dendrimers reflect the

8
tendency of an a polar solvent (poor solvent) to induce a higher molecular density in
the core region due to back-folding (intramolecular polar interactions), and lower
molecular density at the surface. In polar solvents the dendrimer arms are solvated and
the molecular density at the dendrimer surface is increased (M. Ballauff).
A microenvironment can arise in the dendrimer core as a consequence of limited
diffusion of solvent molecules into the dendrimer. As an example, dendrimers
dissolved in polar solvents such as aqueous media can have a very a polar interior
(unimolecular micelle) allowing organic molecules to be encapsulated and carried in
aqueous media. This property of dendrimers makes this class of molecules very well-
suited as carriers of various bioactive substances.

4.3-Properties of dendrimers in biological systems:

Biological properties of dendrimers are crucial because of the growing interest in

using them in biomedical applications. “Cationic” dendrimers (e.g., amine terminated
PAMAM and poly(propyleneimine) dendrimers that form cationic groups at low pH)
are generally haemolytic and cytotoxic.
Their toxicity is generation-dependent and increases with the number of surface
groups (Roberts et al).
PAMAM dendrimers (generation 2, 3 and 4) interact with erythrocyte membrane
proteins causing changes in protein conformation. These changes increase with
generation number and the concentration of dendrimers. Anionic dendrimers, bearing
a carboxylate surface, are not cytotoxic over a broad concentration range (Malik et al).

4.3.1-The significance of multivalency in biological interactions:

Multivalent interactions can be found throughout nature, ranging from the divalent
binding of antibodies and many biological receptors to the multimillion-valent
interactions of a Gecko’s foot hair (K. Autumn et al). Multivalency has been shown to lead
to a strongly increased activity compared to the corresponding monomeric interaction.
This synergistic enhancement of a certain activity e.g. catalytic activity or binding
affinity from a monomeric to a multimeric system, is generally referred to as the
‘cluster’- or ‘dendritic’ effect ( W. B. Turnbull and J. F. Stoddart),( P. H. Ehrlich ),( A. F. Habeeb ),( J. Lundquist et
al).
The dendritic effect is attributed to a co-operative effect in a multivalent system
leading to a larger increase in activity than expected from the valency of the system
(i.e. additive increase).
The multivalent interactions provide:
–tight binding from rather low-affinity binding of single ligands,
–a possibility of utilizing low-affinity ligands in a new arrangement to cope with an
evolutionary new binding partner,
–more efficient cell-cell interactions mediated by multiple interactions.
Factors that play a role in the binding of dendrimeric multivalent ligands include
obviously the geometry of the multimerically presented ligands and the flexibility of
their attachment to e.g. a dendrimer,(Fig. 17) (J. B. Corbell et al).

4.3.2-Biocompatibility of dendrimers:

In order to apply dendrimers as tools for drug design or as drug delivery devices in
vivo, they have to fulfill several biological demands of crucial importance.

9
The dendrimers should be:
– non-toxic;
– non-immunogenic (if not required e.g. for vaccines),
– able to cross biobarriers such as, intestine, blood-tissue barriers, cell membranes,
– able to stay in circulation for the time needed to have a clinical effect,
– able to target to specific structures.

5-Applications:

5.1-Physical association and encapsulation of drugs:

Active pharmaceutical ingredients (APIs) can physically interact with dendrimers

through either encapsulation into void spaces (nanoscale container) or association
with surface groups (nano-scaffolding) or a mixture of both. Driving forces for these
interactions are hydrogen bonding, van der Waals interactions, and electrostatic
attraction between opposite charges on dendrimers and APIs. Small organic molecule
drugs often are encapsulated into the dendrimers’ interior void space, while larger
(bio)molecules preferably adsorb onto the dendrimer surface.
The following listing of drugs physically associated with dendrimers provides an
overview of the breadth of the dendritic platform to serve as drug carriers:
Camptothecin, Cisplatin, Paclitaxel, Diclofenac and mefenamic acid, Doxorubicin,
Etoposide, 5-Fluorouracil, Ibuprofen, Indomethacin, Ketoprofen, and others.
Research activities are centered on three main classes of drugs: anticancer, anti-
inflammatory, and antimicrobial drugs,(Fig.18).

5.2-Chemical conjugation of drugs to dendrimers (prodrug

approach):

Physical interactions between dendrimers and APIs have specific features:

1. They leave the APIs unaltered, and therefore, provide a less challenging
regulatory path forward.
2. They are easy to establish but provide limited control over release kinetics.
3. They often allow only limited drug loading, resulting in rather poor drug-to-
dendrimer ratios.
Alternatively, drugs can be conjugated to dendrimers via chemical bonding.

There are three main pathways to create these prodrugs:

1. Direct conjugation of drugs to the dendrimer surface.

2. Conjugation via a linker molecule if the drugs do not carry the desired
functional group for direct conjugation or if the linker molecules are needed to
modify solubility profiles or release kinetics, or reduce congestion of drug
molecules on the dendrimer surface, allowing a higher degree of conjugation.
3. Drug molecules can become an integral part of the dendritic carrier that is
released through certain triggering events at the desired location, e.g., a tumor
site.

10
Added benefits of the prodrug approach consist in modified pharmacokinetics and
pharmacodynamics.

5.3-Drug delivery properties:

Dendrimers, which are capable of interacting specifically with cancerous tumor tissue,
are an excellent option as a drug transporter within the body. Various polymers and
other highly branched structures lack consistency of functional groups,
monodispersive nature, and uniform molecular weight distribution. Dendrimers also
have an exceptionally high drug loading capacity, which provides a greater
accumulation of drug at the tumor site. Considering that dendrimers can be prepared
with a predetermined, specific number of monomers and polymer branches, as well as
peripheral functional group specific, they are the ideal macromolecule to enter the
highly permeable vasculature of tumor sites and remain localized at the site to deliver
an immense amount (within cytotoxic levels) of drug to the specific tissue . The
surface of dendrimers can be modified with functional groups so that drugs will be
physically entrapped, encapsulated, or conjugated by covalent bonds, ionic
interactions, or hydrogen bonds. With the increase of molecular weight of the drug,
due to the dendrimer-drug interaction, the hydrodynamic volume increases causing
longer circulation time and slower elimination of drug so cytotoxicity levels are
lowered and dosage can be decreased. Lastly, dendrimers have the ability to solubilize
some insoluble anti-cancer drugs. Increased solubility results in higher loading
efficiency, which prevents nonspecific interactions and negative side effects of the
drug.{1}

5.4-Dendrimers as drug delivery devices:

The research in dendrimer mediated drug delivery has mainly been focused on the
delivery of DNA drugs (genes or gene inhibitors) into the cell nucleus for gene or
anti-sense therapy, and numerous reports have been published on the possible use of
unmodified amino-terminated PAMAM or PPI dendrimers as non-viral gene transfer
agents, enhancing the transfection of DNA into the cell nucleus.
It has been found that partially degraded (or fragmented) dendrimers are better suited
for gene delivery than the complete dendrimers , and a fragmentation (activation) step
consisting of hydrolytic cleavage of the amide bonds is needed to enhance the
transfection efficiency.
In comparison to the intact dendrimers, the partially degraded dendrimers have a more
flexible structure (fewer amide bonds) and form a more compact complex with DNA,
which is preferable for gene delivery by the endocytotic pathway.
The unmodified amino-terminated dendrimers transport the DNA to the cell
membrane and may help in the transfection process by disruption of the cell
membrane. The transfection of free DNA will be hampered by electrostatic repulsion
between the negatively charged phosphate groups in the DNA backbone and the
negatively charged cellular membrane.
Tumors, obtain specific properties (increased vasodilatation, vasculature, etc.), can be
combated with a new drug delivery system. The use of monodispersive, polymeric
branched dendrimers as an anti-cancer drug delivery system has shown many
promising results. Dendrimers can be developed with specific properties, which allow

11
for targeting and releasing drugs at the target tumor site. This ultimately leads to lower
dose rates, lower cytotoxicity levels, and higher drug efficiency.{2}

5.5-Folate conjugated dendrimers:

Dendrimers can be conjugated with a ligand specific for targeting tumor sites. Ligands
can be recognized by the appropriate cell surface receptor and thus internalized where
it can deposit the therapeutic agent. One such ligand being tested for use is folate.
Folate appears to be an excellent ligand because many cancerous tumor cells over
express folate receptors , As the folate receptor is over-expressed in cancer cells, these
folic acid derivatised dendrimers are taken up by cancer cells preferentially to normal
cells, making these dendrimers well-suited for the cancer specific drug delivery of
cytotoxic substances. Very recently, folate modified PAMAM dendrimers have been
successfully used as carriers of boron isotopes (10B) in boron neutron-capture
treatment of cancer tumors.
Studies have shown that by using the folate conjugated dendrimer encapsulated with
methotrexate (antifolate drug), there has been better tumor targeting potential, which
decreases tumors to a greater degree compared to the free drug.{1}

5.6-Vectors, in gene therapy:

Dendrimers can act as carriers, called vectors, vectors transfer genes through the cell
membrane into the nucleus. Currently liposomes and genetically engineered viruses
have been mainly used for this. PAMAM dendrimers have also been tested as genetic
material carriers. They are terminated in amino groups which interact with phosphate
groups of nucleic acids. This ensures consistent formation of transfection complexes.
A transfection reagent called "SuperFect" consisting of activated dendrimers is
commercially available. Activated dendrimers can carry a larger amount of genetic
material than viruses. SuperFect–DNA complexes are characterized by high stability
and provide more efficient transport of DNA into the nucleus than liposomes. The
high transfection efficiency of dendrimers may not only be due to their well-defined
shape but may also be caused by the low pK of the amines (3.9 and 6.9). The low pK
permit the dendrimer to buffer the pH change in the endosomal compartment.{3}

5.7-Glycodendrimers:

Glycodendrimers find use as a targeted anticancer drug vehicle because of the

relationship of glycosylation on cancer cell surfaces. This leads to the over expression
of specific antigens that serve as targets for immune recognition by interacting with
lectin-like receptors present on immune cells. Glycodendrimers can be used in a
similar manner by being introduced to the tumor site and acting as an antigen to
promote the accumulation of monoclonal antibodies, which then selectively affect
tumor cells. Glycodendrimers closely resemble the natural carbohydrate ligands,
which add to the selective localization of the drug to the specific site. As with other
dendrimers, the drug is circulated longer and delivered with greater efficiency. The
formation of these dendrimers can occur in three ways:
• Fully coated dendrimers with carbohydrates,
• Carbohydrate moieties at the periphery, and
• Carbohydrates at the center of the dendrimer.{1}

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5.8-PEGylated dendrimers:

PEG modification on the surface of the dendrimers is receiving more attentions

because it increases water solubility and biocompatibility of the dendrimers for
drug delivery.{4}
Polyethylene glycol (PEG) dendrimers are non-toxic and avoid detection by the
immune system, protein adsorption, and show tissue specificity. By inserting drugs
into PEGylated dendrimers, there is an improved efficiency of delivery to the tumor
site of that drug and an increase in the half-life because of the prevention of
deterioration due to non-specific interactions. This increase in stability ultimately
results in the reduction of doses, increased tumor selective uptake, and reduced
allergic responses. Using PEGylated dendrimers also has the advantage of reduced
cytotoxicity and immunogenicity.
A drawback in other dendrimer system. Kojima et al. reported that PEG solubility is
affected by varying the chain length. Studies were done with varying chain sizes and
by encapsulating anti-cancer drugs, methotrexate, and doxorubicin. The results of the
study showed that with an increase chain length, there was an increase in
encapsulation efficiency and dendrimer generation was seen.{1}

5.9-Peptide dendrimers:

Peptide dendrimers consist of amino acids as the core molecules with the outer
polymers made up of peptide moieties or amino acids. These dendrimers are effective
as drug vehicles because peptides can trigger apoptosis and inhibit the growth of
epithelial tissues. A common type of peptide dendrimer is the L-lysine dendrimer,
which has shown to be a molecular inhibitor of angiogenic factors. Also, the L-lysine
dendrimer has shown to stimulate an immune response creating antibodies specific
against tumors. Other methods in which peptides act in a medicinal way are the
obstruction of cell cell interactions and the prevention of cell adhesion.{1}

5.10-PH sensitive dendrimers:

Dendrimers of this type generated to exhibit specific properties which allow them to
be small enough to enter normal vessels, as well as being of optimal size to enter the
tumor through the increased pore size. Once inside the cell or tumor site there is a
change in pH, which then affects the conformation of the dendrimer . One method of
how pH sensitive dendrimers are used for drug transport is that at normal
physiological pH (7.4) the terminal amine groups on a polypropylene imine dendrimer
are not protonated and the branches converge onto the center multifunctional molecule
or drug. Upon entering the microenvironment of the tumor site, pH drops to a more
acidic level and the terminal amine groups are now protonated and repel one another.
This stimulates the release of the drug at the tumor site by the opening of the branches
outward. pH sensitive dendrimers can also be fitted with functional groups on the
surface of the dendrimer. By adding various functional groups such as an amine to the
dendrimer surface, release of drugs occurs when the functional group protonates.
Additionally, because the release of the drug is pH dependent, the degree of side
effects and cytotoxicity decreases.{1}

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5.11-Dendrimers in Topical Drug Delivery:

Surface modifications of dendrimers have been used as molecular-carrying systems.

For example, a keratolytic or anti-acne agent was complexed with a carrying molecule
such as a dendrimer containing free amino groups to obtain cosmetically acceptable
formulations for treatment of acne vulgaris. In another example of a dendrimer-
molecule conjugate system, coupling of amino butadiene with an amine-rich dendritic
molecule provided advantageous UV-absorbing capabilities to the final product. This
high-molecular-weight dendrimer-amino butadiene-complexed molecule allowed ease
in formulating a clear sunscreen composition without developing high-viscosity gels,
which in turn provided ease of application to the skin. Because of the high molecular
weight of the resulting molecule, it was nonpenetrating into the skin, which would
minimize risk of irritation or sensitization reactions while acting as a UV-light
absorber when applied on the skin's surface. In another application, amine-terminated
cationic dendrimers have been used in personal-care cleansing compositions as
mildness agents. Linear cationic polymers used as mildness agents usually precipitate
in the presence of anionic surfactants, which reduces their lathering, skin
conditioning, or cleansing effects. Dendrimers, on the other hand, are capable of
interacting favorably and can bind with anionic surfactants in the composition to
remain dispersed in salt solutions. This interaction of cationic dendrimers with skin-
irritating anionic surfactants could potentially be used for reducing the skin irritation
potential of cosmetic formulations containing harsh anionic surfactants.{5}

5.12-Dendrimers in transdermal delivery:

Dendrimers have found recent applications in novel transdermal delivery system,

providing benefits such as improved drug solubilization, controlled release, and drug-
polymer conjugates (pro-drugs). The viscosity-generation-number property of a
dendrimer solution allows for ease of handling of highly concentrated dendrimer
formulations for these applications. Dendrimers have been shown to be useful as
transdermal drug delivery system for nonsteroidal anti-inflammatory drugs (NSAIDs),
antiviral, antimicrobial, anticancer, or antihypertensive drugs. PAMAM dendrimers
have been studied as carrier transdermal systems for the model NSAIDs: ketoprofen
and diflunisal. It was found that the PAMAM dendrimer-drug formulations showed
increased transdermal drug delivery compared with formulations lacking dendrimers.
In vivo studies in mice showed prolonged pharmacodynamic responses and 2.73-fold
higher bioavailability over 24 h for certain dendrimer-containing drug solutions.{5}

5.13-Dendrimers as ophthalmic vehicles:

The majority of topically applied ocular drug-delivery systems are formulated either
as solutions, ointments, or suspensions and suffer from various disadvantages such as
quick elimination from the precorneal region, poor bioavailability, or failure to deliver
the drug in a sustained fashion. Several research advances have been made in ocular
drug delivery systems by using specialized delivery systems such as polymers,
liposomes, or dendrimers to overcome some of these disadvantages. Ideal ocular drug-
delivery systems should be nonirritating, sterile, isotonic, biocompatible, and
biodegradable. The viscosity of the final product should be optimized so that the
dosage form does not run out of the eye. Dendrimers provide solutions to some
complex delivery problems for ocular drug delivery.

14
In particular, the cationic dendrimers are of interest for their application in the ocular
topical pathway since they exhibit high interaction with the mucins of the corneal
epithelium which are loaded negatively in physiological conditions by the presence of
sialic groups. This electrostatic interaction converts these dendrimeres in
mucoadhesive compounds able to generate an increase in the contact period of the
pharmaceutical form (and the drug that it contains) on the surface of the eye. In
addition, the cationic nature of these dendrimeres also induces electrostatic interaction
with proteins of the epithelial intercellular unions, generating a temporary
reorganization of these structures and an increase of their para cellular permeability.
These properties are attractive for the use of these polymers as promotional agents of
the penetration of active substances through the cornea.{6} In the New Zealand albino
rabbit model, the residence time of pilocarpine in the eye was increased by using
dendrimers with carboxylic or hydroxyl surface groups. These surface-modified
dendrimers were predicted to enhance pilocarpine bioavailability.{5}

5.14-Dendrimer drugs:

A-Dendrimers as antiviral drugs:

In general, antiviral dendrimers work as artificial mimics of the anionic cell surfaces,
thus the dendrimers are generally designed having anionic surface groups such as
sulfonate residues or sialic acid residues, which are acidic carbohydrates present at the
mammalian cell surface. In other words, the dendritic drug competes with the cellular
surface for binding of virus, leading to a lower cell-virus infection probability.
Polylysine dendrimers modified with naphtyl residues and having sulfonate surface
groups have been found to be useful as viral inhibitors for Herpes Simplex virus in
vitro. PAMAM dendrimers covalently modified with naphthyl sulfonate residues at the
surface, giving an polyanionic surface, also show antiviral activity against HIV. Also
here the dendrimer drug works as an inhibitor for early stage virus/cell adsorption and
at later stages of viral replication by interfering with the reverse transcriptase and/or
integrase enzymes.{7}

B-Dendrimers as antibacterial drugs:

In contrast to the antiviral dendrimers, the antibacterial dendrimers generally contain

cationic surface functionalities such as amines or tetra alkyl ammonium groups. The
general mode of action of the antibacterial dendrimer is to adhere to and damage the
anionic bacterial membrane, causing bacterial lysis.
PPI dendrimers where the surface has been functionalized with tertiary alkyl
ammonium groups have shown to be very potent antibacterial biocides against Gram
positive and Gram negative bacteria.
Polylysine dendrimers having mannosyl surface groups have been shown to inhibit
adhesion of E. coli to horse blood cells in a haemagglutination assay, making these
structures promising as antibacterial agents.{7}

5.15-Dendrimers as protein denaturants:

Certain types of dendrimers act as chaotropes i.e. water structure perturbing solutes,
lowering the dielectric constant and the viscosity of water; disordering the regular
water structure by reorganizing water molecules at the dendrimer surface. As with

15
other chaotropes, this leads to hydrophobic interactions being disfavored which, in
turn, is highly destabilizing for most protein tertiary structures (denaturation).
Classical examples of chaotrophic salts are MgCl2, urea, guanidinium chloride,
sodium thiocyanate, guanidinium thiocyanate at high concentrations and other
chaotropes include polarity-decreasing, water miscible organic solvents such as
acetonitrile, propanol and methanol. Generally, chaotropes will serve to denature and
solubilise proteins, which is useful for example in solubilising protein aggregates as
are often encountered when expressing proteins in heterologous expression systems
(inclusion body formation) and when extracting certain types of membrane proteins.
Dendrimers, being compact, large polyionic substances have the physicochemical
properties needed to make them potential chaotropes/protein denaturants.
A striking example of this was cationic dendrimers were used for the solubilisation of
prion protein aggregates. Prion proteins are able to attain a pathogenic
structure/conformation in which they can cause mortal diseases called spongiform
encephalopathy's, including mad cow disease and Creutzfeldt-Jakob disease. These
deadly conformers are characterized by their tendency to form very insoluble
aggregates, which are found in the brains of affected individuals. Such aggregates are
soluble only in solvents containing both detergent and denaturant (typically 6 M
guanidinium chloride), however it was shown that such aggregates can be solubilised
by cationic dendrimers, such as PEI-, PPI- and PAMAM dendrimers, higher
generation ( > G3) dendrimers being the most efficient and influenced by the number
of surface amino groups. PAMAM dendrimers having hydroxyl groups at their surface
(PAMAM-OH)and linear polymers had no or very minor effects. The effect was seen
at surprisingly low concentrations (7 mg ml21 or below) on aggregate producing
neuroblastoma cells and took place with no cytotoxicity.{7}

5.16-Dendrimers in vaccines:

It is well-established that small molecular weight substances (e.g. peptides) are not
very immunogenic. i.e. no or a weak immune response (including antibody formation)
is induced upon their injection into a recipient host. However, this problem can be
overcome by increasing the molecular weight of the substance in question either by
polymerization or by coupling it to a multifunctional, high molecular weight carrier,
traditionally a naturally derived protein. For the preparation of highly defined,
reproducible immunogens e.g. for human vaccine uses, other types of carriers are
highly desirable and in this respect, dendrimers have emerged as useful since they can
act as multivalent and well defined carriers for antigenic substances by coupling of
antigen molecules to the surface functional groups of the dendrimer.
Examples of dendrimer–peptide compounds being used for vaccine and immunization
purposes include the multiple antigenic peptide (MAP) dendrimer system pioneered
by Tam and coworkers, which can be synthesized with defined mixtures of B and
T-cell epitopes, either synthesized by stepwise peptide synthesis on the branches of
the MAP or by segment coupling of peptide fragments by various methods.

16
6-Conclusion:
Dendritic polymers are expected to play a key role as enabling building blocks for
nanotechnology during the 21st century. The controlled 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 amongst 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.

17