International Journal of Current Chemistry : Vol. 1, No.

4, October-December 2010 195

REVIEW

Hyaluronan-mediated CD44 Receptor Cancer Cells

Progression and the Application of Controlled
Drug-delivery System
Gul-e-Saba1, A. Abdah2, M.A. Abdullah1
1
Department of Chemical Engineering, University Technology PETRONAS,
Bandar Seri Iskandar, 31750, Perak, Malaysia
2
Department of Biomedical Sciences, University Putra Malaysia,
Serdang, 42400, Selangor, Malaysia

ABSTRACT
Hyaluronan (HA) is a major component of extracellular matrix (ECM) and
main ligand of CD44 receptor cells. CD44 belongs to multifunctional transmembrane
protein family expressed in both normal and tumor cells. HA is expressed more in
cancer cells as compared to normal cells. HA binding to CD44 mediate oncogenic
signaling, metastasis and cytoskeleton activation leading to tumor invasion and growth
of tumor cells. HA-CD44 binding and Cytoskeleton signaling pathways are important
targets for anti-cancer drug development to curb tumor invasion and metastasis. A
polymer drug delivery system such as Dendrimers can possibly combat deadly
cancer. Globular macromolecular versatile polymer having multivalent, monodisperse
and highly branched carrier molecule will play a major role in targeted control drug
delivery against cancer.
Keywords : Hyaluronan, CD44 receptor, dendrimers, oncogenic signaling, drug
delivery.
(Recieved: 08-06-2010; Accepted: 15-10-2010)

1. INTRODUCTION
Cancer is uncontrolled growth, invasion and metastatic behavior of group of cells. Most cancers
arise from a tumor. Cancer affects people at all ages with the risk for most types increasing with age
[1]. Cancer has caused about 13% of all human deaths in 2007 [2]. In cancer cells, CD44 receptor is
expressed more as compared to normal cells. Hyaluronan (HA) is however found to be secreted by
various normal and cancer cells. Any interaction between CD44 receptor and hyaluronan ligand can

M.A. AdbdullahDepartment of Chemical Engineering, University Teknology

PETRONAS, Bandar Seri Iskandar, 31750, Tronoh, Perak, Malaysia. IJCC
E-mail : azmuddin@petronas.com.my 195
196 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

mediate both cellcell and cellextracellular matrix interactions and has been shown to be important
in a variety of physiological and pathophysiological processes including tumor metastasis, wound
healing and leukocyte extravasation at the sites of inflammation[3]. This review article attempts to
look at the role of CD44 as a cell surface signaling protein in various tumor cells, HA-CD44 interaction
and signaling pathway in tumor cells via various cytoskeleton proteins leading to tumor cell growth,
adhesion, and metastasis. The role of dendrimer in controlled targeted drug delivery is also reviewed.
2. MULTIFUNCTIONAL CD44, A CELL SURFACE SIGNALING PROTEIN
The multifunctional CD44 molecule plays distinct functions in cell adhesion, signal transduction,
and cell-cell communication. CD44 receptor protein binds to extracellular matrix components [4],
thus mediates homotypic cell-cell adhesion [5]. Besides, CD44 can trigger heterophilic adhesion
events such as the interaction between leukocytes and endothelial cells [6]. CD44 also acts as a
powerful signaling molecule through tyrosine kinases [7], proteins belonging to the src family, having
a close association with cytoplasmic regions of CD44 upon ligand binding, tyrosine kinases mediate
cytoskeleton protein activation through phosphorylation trigger. These signaling events lead to
activation of the NF-kB/I-kBa pathway [8] and induction of proinflammatory chemokine expression
in macrophages [9]. Furthermore, multifunctional CD44 also perform various signaling and cellular
communication functions. HA mediated CD44 signaling pathways in tumor cells have been studied
[6,9,10]. Molecular size of hyaluronan (HA) fragments is found critical for recognition and signal
transduction via CD44. In addition, binding of modified HA ligands can act as a switch between
adhesive and signaling functions [10].
CD44 Structural Features and Ligands
CD44 consists of three domains- extracellular, cytoplasmic and transmembrane domain, as
shown in Fig 1. There is also ligand binding site and cytoskeleton binding protein site on CD44

Fig 1 Schematic representation of CD44 molecule.

International Journal of Current Chemistry : Vol. 1, No. 4, October-December 2010 197

molecule. Alternate splicing occurs in extracellular domain because of the presence of variable
spliced exon (v1-v10) to produce various CD44 isoform. Functional features of CD44 receptor
depends on cellular environment, post translation modification and also on cell specific isoforms
[11,12,13]. CD44 also belongs to hyaloadherin family or link protein super family, LPSF [14,15].
LPSF, is also termed proteoglycans, because of the addition of glycosaminoglycans that may be
keratin sulfate, chondroitin and heparin [16]. lymphatic vessel endothelial (LYVE-1), another
hyaluronan-binding protein having close homology to CD44 (40% matching sequence), is also known
as a new member of this family [17]. Several ligands have been identified for CD44 protein including
HA and chondroitin sulfate [18,19,20], collagen and the heparin-binding domain of fibronectin
[21,22]. The role of the cytokine osteopontin as a potential CD44 ligand remains debatable [23,24].
Nevertheless, it has been firmly established that CD44 is the major cell surface receptor for HA
[20]. Many of the functions of CD44 in cell adhesion and activation can be attributed to CD44
binding [10]. HA mediated CD44 activation can be critical for tumor progression.
HA Binding and Regulation
HA is a polymeric glycosaminoglycan and a major component of the extracellular matrix
[25,26]. It consists of repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units[27].
Fig. 2 shows the structure of an HA trisaccharide [28]. This trisaccharide consists of two D-glucuronic
acid units and N-acetyl-D-glucosamine and contains two distinct glycosidic bonds (13 and 1
4). The size of HA fragments recognized by CD44 is very critical for adhesive and signaling functions
[10]. At least an HA hexasaccharide is required for effective recognition by CD44 [29,30] but a
decasaccharide unit has been reported capable of replacing polymeric HA bound to CD44, expressed
on keratinocytes [31]. Furthermore, CD44 play an important role in signalling and activation of
immune system especially during tissue damage or extracellular matrix degradation [32,33].

Fig 2 Structure of a small hyaluronan fragment [50].

HA binding activity mainly resides in link module. However, some mutations or deletions in
all domains of CD44 are found to affect HA binding [34]. Glycosylation of CD44 can be critical for
HA binding, though it is not completely understood. In E.coli an active form of CD44 is expressed
upon HA binding suggesting that glycosylation may not be critical for HA recognition [35]. However,
treatment with inhibitor (tunicamycin, an inhibitor of N-linked glycosylation or genetic disruption
mutation or deletion of N-linked glycosylation sites in CD44) on CD44-expressing cell lines inhibits
198 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

HA binding [36]. Reduced level of N- and O-linked glycosylation often results in improved HA
binding [37,38].These prove that modification (glycosylation) can affect the ligand-receptor (HA-
CD44) binding, but further research is needed to better understand the role of CD44 modification
(glycosylation) on HA (major ligand) binding.
The Role of HA- CD44 Interaction in Tumor Cells
Tumor invasion and metastasis are the primary causes of sickness in patients diagnosed with
solid tumors such as breast cancer, ovarian cancer and squamous cell carcinomas [39,40,41]. Ligands
play mediating roles in various oncogenic signals. Oncogenic signaling and cytoskeleton functions
are directly involved in tumor cell growth, invasion, and metastasis [42,43]. Ligand binds to specific
receptor and after binding receptor becomes activated, the step by step regulation ensues. HA is
synthesized by specific HA synthases and digested into various smaller-sized molecules by various
hyaluronidases [27,44,45]. HA is enriched in many types of tumors [46,47]. The concentrations of
HA are reportedly higher in malignant tumors as compared to benign or normal tissues and in some
tumor types the level of HA is predictive of malignancy [47]. HA interacts with a specific cell
surface receptor, CD44, which belongs to a family of multifunctional transmembrane glycoproteins
expressed in numerous cells and tissues including tumor cells and carcinoma tissues [48]. Alternative
splicing occurs in extracellular domain of CD44, due to the presence of variant exons v1-v10 resulting
in a variety of isoforms produced [49]. The CD44 variant (CD44v) isoforms expression has been
found to be closely associated with tumor progression [48].
All CD44 isoforms exhibit HA-binding site in their extracellular domain, and thereby serve as
a major cell surface receptor for HA [51]. It has also been demonstrated that HA is rich in stem cell
niches [52]. CD44 receptor is expressed in both normal and tumor stem cells (displaying unique
ability to initiate normal and/or tumor cell-specific properties) and CD44 has been suggested as one
of the important surface markers for both normal stem cells and cancer stem cells [53]. The fact that
both CD44 and HA are overexpressed at the sites of tumor and HA-CD44 interaction and binding
stimulate tumor progression [48] suggests that HACD44 interaction may be a critical requirement
for tumor progression.
3. HA-CD44 SIGNALING PATHWAYS IN TUMOR CELLS
Fig. 3 shows the HA mediated CD44 signaling pathways in tumor cells. These principally
involve ankyrin-activated pathways and Rho A (GTPases) pathways. HA interaction with CD44
receptor of cancer cell may also result in cancer cell progression via cytoskeleton protein activation
such as the kinases, the guanine nucleotide exchange factors and the AKT signaling molecules.
Ankyrin-activated Pathways
CD44 interacts with several cytoskyleton proteins as well as membrane-bound cytoskeletal
protein such as ankyrin, expressed in various biological systems [54,55]. Ankyrin has three functional
domains:- ankyrin repeat domain (ARD) at N terminal, a spectrin binding domain and C terminal
variable size domain [56]. CD44 binds directly with ankyrin by a highly conserved sequence (almost
70 amino acid sequence) that is present in most of CD44 isoforms [57,58]. More importantly, ankyrin
binding domain of CD44 is necessary for oncogenic signaling [59]. ARD binding domain of ankyrin
has been identified in the binding of both CD44 and Tiam 1, a cytoskeleton protein [49,60]. In
normal cells, protein-protein interaction is tightly regulated. In the case of cancer/tumor cells, there
will be a misregulation in this interaction. Cytoskeleton protein ankyrin, if over expressed, promotes
HA dependent and CD44 mediated migration of tumor cell [61].
International Journal of Current Chemistry : Vol. 1, No. 4, October-December 2010 199

Ankyrin interaction with CD44 results in HA mediated cytoskeleton activation, cell proliferation,
adhesion and migration [54,55,58]. HA binding with CD44 receptor protein promotes recruitment
of several proteins such as ankyrin and inositol-1,4,5-triphosphate (IP3) receptor into specific place
in cell membrane, that is made up of distinct protein and lipid composition known as lipid rafts
during cell signaling pathways [62]. In lipid, raft IP3 receptor binds to CD44, triggering mobilization
of (Ca2+) which leads to cell adhesion and migration. ARD domain of ankyrin is responsible for
binding of IP3 receptor to CD44 in lipid raft, in signal cascade, leading to cell adhesion and
proliferation [62]. Lipid raft acts as a potential target for drug in tumor cell as it mediates the tumor
cell behavior. Any deletion/disruption in lipid raft inhibits the interaction between CD44 and IP3
receptor [63]. Lipid raft is valuable for co-localization of CD44 and ankyrin. This can be a future
target for drug delivery to inhibit the signaling cascades, and also to curb tumor cell invasion and
migration.

Fig 3 HA mediated CD44 signaling pathways in tumor cells.

Fig 3 [Modified from 64]: HA mediated CD44 signaling pathways in tumor cells leads to cell
adhesion, migration, tumor cell growth and invasion. HA bind with CD44 leads to activation in
cytoplasmic domain of CD44. A: Ankyrin (membrane bound cytoskeleton signaling protein) activated
pathway that results in cell adhesion; B: Rho-A (GTPases) activation that promote phosphoinoside-
specific phospholipase C--IP3 (PLC-IP3) production and Ca2+ mobility which further undergoes
Filamin activation (by phosphorylation) through Ca+2/ calmodulin-dependent kinase II and promotes
cell migration and invasion. Rho A is itself activated by Rho GEF LARG factor. B2-1 & B2-II ROK
(Rho binding Kinase) facilitates ECM degradation and actomysin-mediated cell migration,
respectively. However in B2-III AKT Pathway mediated, tumor cell survival and growth occur.
ROK also mediate recruitment of ankyrin to lipid rafts of cell membrane.
200 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

Guanine Nucleotide Exchange Factors (GEF)

A member of Rho subclass of Ras superfamily Rho A, Rac 1 and cdc42 known as GTPases
[65], play active role in signal cascade. GTPases are energy dependent Molecular switches that
transmit oncogenic signals from HA-CD44 to intercellular targets leading to cytoskeletal protein
activation and cell migration process [65,66]. Rho GTPases are activated by various guanine
nucleotide exchange factors (GEFs) [67]. LARG (leukemia-associated Rho guanine nucleotide
exchange factor) has been found as Rho A specific GEF that contain different domains-amino acid
terminal domain (PDZ), regulator of G-protein signaling (RGS) domain, dbl homology (DH) domain
and pleckstrin homology (PH) domain [68]. DH domain of LARG protein play an important role in
Rho A activation depending on GDP/GTP exchange activity [69].
LARG mediated Rho A signaling has been confirmed in the neck and head tumor, as being
induced by HA binding to CD44 receptor of tumor cells [70]. After activation of Rho A, Rho-PLCe
coupled protein (GTP dependent protein) enhance PLCe- mediated IP3 production and the Ca 2+
ions mobilization activate (upregulate) Ca2+/ calmodulin-dependent kinase II (CaMKII) which leads
to phosphorus addition to filamin, a cytoskeletal protein. Phoshorylation (activation) of filamin
causes repelling interaction with actin filament, which activates tumor cell migration [70]. However
change in expression (overexpression) of PDZ domain of LARG abolishes tumor cell migratory
behavior by inhibiting HA-CD44 mediated signaling cascade [70]. The above finding clearly suggests
that LARG may play a vital role in head and neck tumor cells progression.
Besides LARG, there is another guanine nucleotide exchange factor, P115 Rho GEF, which
involves in Rho activation [71]. Similar to LARG, it consists of several functional domains: - RGS
(amino-terminal regulatory of G- protein signaling) domain, DH domain (a dbl homology) and PH
(pleckstrin homology) domain [72,73]. P115 Rho GEF also contains sequence that is homologous
to DH domain of a specific member of Ras superfamily, which exhibits GDP/GTP exchange activity
[74]. In breast tumor cells, P115 RhoGEF is found to be closely associated with CD44. Furthermore,
HA binding with CD44 of breast tumor cells also induces/stimulates P115 RhoGEF mediated Rho A
oncogenic signaling [75] and metastasis.
Rho Binding Kinase
HA-CD44 binding also generates/produces actomyosin mediated/induced membrane motility
and possible role in cell migration via ROK (Rho binding kinase). ROK has been found as a possible
enzyme target for Rho A (Rho GTPases protein), a serine threonine kinase [76,77]. HA-CD44 binding
activates the ROK as well as interacts with Rho A (in GTP dependent manner) performing several
protein activation [75,78]. ROK also plays important role in the binding of CD44 (cytoplasmic
domain) with ankyrin (membrane bound cytoskeletal protein) by phosphorylation [73]. ROK consists
of Catalytic domain (CAT), coiled coil domain, Rho binding domain and PH domain. Furthermore,
it binds to Rho A protein and plays a part in cytoskeleton rearrangement and cell migration [79,77].
More importantly, ROK enzyme phosphorylates myosine phosphatase enzyme and myosine light
chain [80]. This supports suggestion on HA mediated CD44 and cytoskeletal proteins interaction,
leading to oncogenesis in tumor cells and tumor cell migration.
AKT Signaling
Tumor cell growth, cell survival and invasion are also HA-CD44 mediated via AKT signaling
in tumor cells. Rho A mediated ROK activation and Gab-1 mediated phosphatidylinositol-3-kinase
(PI3 kinase) activation play a vital role in AKT signaling in tumor cells mediated by HA-CD44
interaction [75]. Similarly, HA activates PI3 kinase AKT signaling pathway that leads to cell motility
International Journal of Current Chemistry : Vol. 1, No. 4, October-December 2010 201

and survival [71]. It has been studied that in colon carcinoma cells, PI3 kinase is an important player
of cell survival pathway mediated by CD44 [81]. Gab-1 (Grb-2-associated binder-1), a member of
insulin receptor substrate (IRS) family is adaptor molecule and key mediator in regulating oncogenesis
[81]. It activates downstream cellular proteins involved in cell survival, tumor cell growth and cell
invasion.
Acidification of Cell Microenvironment
Major causes of treatment failure are tumor invasion and metastasis in cancer patients [82].
Acidic pH favors ECM degradation as one study has shown that ECM degradation is often associated
with acidification of microenvironment of tumor cells [83]. Studies have supported that mammary
cancer cells have the ability to acidify the extracellular environment more rapidly than the normal
mammary cells [84]. Lactate dehydrogenase is also reportedly capable of acidifying primary tumor
environment [85]. However, a number of studies have been conducted to determine the molecule
that changes the environment (suitable for degradation). Among different molecules, NHE (Na +-H+
exchanger), a ubiquitous protein is found to regulate cellular pH [86]. NHE-1 is a potent isoform
which catalyzes (Na+-H+ ions exchange) and regulates intracellular pH i.e. pHi found in breast tumor
cells [87]. Moreover, NHE-1 isoform serves as a substrate for ROK-enzyme [88]. HA binds to
CD44 leading to NHE-1 activation by phosphorylation and eventually cancer progression by matrix
degradation occurs [89].
Cell Migration
CD44 plays an important role in cell migration and any misregulation of CD44 receptor protein
can contribute to malignancies and disease pathologies. CD44 and MMPs (Matrix metalloproteinase)
can enhance or regulate cell migration. MMPs are a family of endo-peptidases (Zn dependent)
enzyme which involve in degradation of ECM (extracellular matrix) components [90] and enhance
movement of cells. Furthermore, membrane-type 1 matrix metalloproteinase (MT-1-MMP) cleaves
extracellular domain of CD44 molecule (about 70 kDa). This cleaved soluble molecule interferes
HA binding with membrane bound CD44 during immune activity [91]. Possible role of CD44 in cell
migration is that it can regulate secretion and activation of MMP-2 [92] along with providing support
to MMP-9 (protolytically active form) on the surface of tumor cell lines [93].
4. DENDRIMER AS A NOVEL POLYMER AND DELIVERY AGENT
Dendrimer belongs to a group of polymers, a versatile globular macromolecule with well
defined physical, chemical and biological characteristics. It possesses a highly branched 3D structure
consisting of molecular chains branching out from a common centre without any entanglement between
molecules. Dendrimers have often been referred to as the Polymers of the 21st century. Dendrimer
chemistry was first introduced in 1978 by Fritz Vogtle and coworkers [94], where the first cascade
molecules were synthesized. In 1985, the first family of dendrimers was synthesized by Tomalia
and co-workers [95]. The word dendrimer originated from the Greek word dendron, for tree,
and meros, for part. Due to multifunctional characteristics, dendrimers have stimulated wide interest
in various fields of chemistry and biology especially in applications such as gene therapy,
chemotherapy and drug delivery.
Structure of Dendrimer
Dendrimers are built from a starting atom, such as nitrogen, to which carbon and other elements
are added by a repeating series of chemical reactions that produce a spherical branching structure.
202 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

As the process repeats, successive layers are added, and the sphere can be expanded to the size
required. The result is a spherical macromolecular structure whose size is similar to albumin and
hemoglobin, but smaller than such multimers as the IgM antibody complex. As shown in Fig 4,
dendrimers possess three distinguished architectural components [96]:
An initiator core
Interior layers (generations) composed of repeating units, radially attached to the interior
core
Exterior (terminal functionality) attached to the outermost interior generations
The components of a dendrimer structure include:
i) Generation: This is a hyper branching region, going from the centre of the dendrimer
towards the periphery, resulting in homo-structural layers between the focal points (branching
points). The number of focal points from the core towards the surface is the generation
number. Dendrimer having five focal points from the centre to the periphery is denoted as
the 5th generation dendrimer, abbreviated a G5-dendrimer. The core part is sometimes
denoted generation zero, or G0 representing null generation.
ii) Shell: This is a homo-structural spatial segment between the focal points, the generation
space. The outer shell is the space between the last outer branching point and the surface.
The inner shells are generally referred to as the interior.
iii) Pincer: The outer shell consists of a varying number of pincers created by the last focal
point before reaching the dendrimer surface. In poly (propylene imine) (PPI) and Poly
(amido amine) (PAMAM) dendrimers, the number of pincers is half the number of surface
groups (because in these dendrimers the chain divides into two chains in each focal point).
iv) End-group : This is also referred to as the terminal group or the surface group of the
dendrimer. Dendrimers having amine end-groups are termed amino-terminated dendrimers
[97].

Fig 4 Schematic representation of dendrimer molecule including (i) Initial Core

(ii) Generations (iii) Exterior (Terminal functionality) attached to the last generation.
International Journal of Current Chemistry : Vol. 1, No. 4, October-December 2010 203

Dendrimer Synthesis
Dendrimers are usually synthesized by either using divergent or convergent approach [98]. In
the divergent methods, dendrimer branches grow outwards from a multifunctional core molecule.
The core molecule binds with monomer molecules containing one reactive and two dormant groups
giving the first generation of dendrimer. After first generation, the periphery of the molecule is
activated for reactions with more monomer molecules to form second periphery. The process is
repeated again and again for several generations to attain desirable generation (Fig. 5A). This approach
is successful for the production of large quantities of dendrimers. However side reactions and
incomplete reactions of the end groups may occur that lead to structure defects. To prevent this,
large excess of reagent is required. This causes some purification difficulties of the final product.
The convergent methods are developed as a response to the weaknesses of the divergent synthesis
[99]. In the convergent approach, the dendrimer is constructed stepwise, starting from the end groups
and then multifunctional core molecule attached. After the synthesis of arms, called dendrons, they
are attached to a multifunctional core molecule (Fig. 5B).

Fig 5 A. The divergent growth method, B. The convergent growth method [100]

Novel Polymer: Biological Features

Dendrimers constitute ubiquitous type of precisely defined polymers [101]. Unlike linear
polymers, they are monodisperse macromolecules. Size and molecular mass can be easily tuned/
controlled during the synthesis by varying the generation number. Size also determines the toxicity
of particular dendrimer such as PPI and PAMAM dendrimer [102,103]. As in the case of PAMAM
dendrimer, cytotoxicity increases with generation for both full-generation cationic dendrimer and
half-generation anionic intermediates [103-105] which can be reduced by modifying surface amine
group with neutral or anionic moieties [106]. Dendrimer solutions are significantly less viscous than
linear polymers [107] as the former dendrimer exhibits a form of tightly packed ball increasing
intrinsic velocity, the molecular mass goes through a maximum of fourth generation and then begins
to decline [108] which is unlikely in linear polymer. Dendrimer toxicity can depend on surface
charge and more on functional group. Cationic (surface) charges are in general more toxic and it has
been proposed that primary amines are relatively more toxic than secondary or tertiary amines. A
concentration dependent tendency to cause haemolysis and changes in erythrocyte morphology has
been linked to the presence of NH2 groups [106]. However PPI dendrimers with diaminobutane
(DAB) and diaminoethane (DAE) cores do not show generation dependence for the haemolytic
effect [109-110].
204 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

Dendrimers are globular in shape containing internal cavities for any type of guest molecule
(bio-particle). Drugs/ guest molecule can either be attached to the periphery of dendrimer molecule
or trapped between the molecular chains (Figure 6). The most important one is the attached drug
macromolecule in the interior to enhance its properties. The guest molecules to be entrapped depend
on its shape and the architecture of the box and its cavities. Meijer and co-workers have described
the experiment in which they had trapped four to eight to ten molecules in one dendrimer [111-112].
The small molecules, like rose bengal or p-nitrobenzoic acid are trapped inside the dendritic box
of poly (propylene imine) dendrimer with 64 branches on the periphery. A shell is formed on the
surface of the dendrimer by reacting the terminal amines with an amino acid (L-phenylalanine) and
the guest molecules are stably encapsulated inside the box. Hydrolysing the outer shell could liberate
the guest molecules, which could be done by particular factors such as pH or any other condition/
micro-environment.

a) b)
Fig 6 a) Drug attached to the periphery, b) Drug/guest molecule entangled inside

The presence of many chain-ends is responsible for high solubility and miscibility and for
high reactivity [107]. There are many substances, which have a strong therapeutic activity. However,
due to their lack of solubility in desired solvents, they have not been used for therapeutic purposes.
In a solubility test with tetrahydrofuran (THF) as the solvent, the solubility of dendritic polyester is
found remarkably higher than that of analogous linear polyester. A marked difference is also observed
in chemical reactivity. Dendritic polyester is debenzylated by catalytic hydrogenolysis whereas linear
polyester is unreactive. Similarly, lower generation dendrimers which are large enough to be spherical
but do not form a tightly packed surface, have enormous surface areas in relation to volume (up to
1000 m2/g) [113].
Water soluble dendrimers are capable of binding and solubilizing small acidic hydrophobic
molecules with antifungal or antibacterial properties. Dendrimers having a hydrophobic core and a
hydrophilic surface layer have been termed unimolecular micelles. Unlike traditional micelles,
dendrimers do not have a critical micelle concentration. This characteristic offers the opportunity to
solubilize poorly soluble drugs by encapsulating them within the dendritic structure at all
concentrations of dendrimer. A hydrophilichydrophobic core-shell dendrimer with PAMAM interior
and long alkane chain exterior is shown to bind 5-flurouracil, a water-soluble anti-tumor drug [114].
International Journal of Current Chemistry : Vol. 1, No. 4, October-December 2010 205

5. DENDRIMER APPLICATIONS
Drug Delivery
Versatile polymer, macromolecular highly branched architectures containing a number of
controlled terminal groups play a promising role in biomedical applications [115-116] and more as
drug carriers [114]. Drug molecules can be loaded both in the interior of the dendrimers as well as
(exterior) attached to the surface groups [117]. Dendrimers can function as drug carriers either by
encapsulating drugs within the dendritic structure, or by interacting with drugs at their terminal
functional groups via electrostatic or covalent bonds (pro-drug) [114].
Delivery of Anticancer Drugs : Dendrimer have shown positive results regarding cytotoxicity
and systemic circulatory half-life (72 h). By improving the drug properties such as solubility and
plasma circulation time, the polymeric carriers can facilitate the passive targeting of drugs to solid
tumors resulting in accumulation of macromolecules in tumor tissue a phenomenon termed the
Enhanced Permeation and Retention (EPR) effect [114]. The cytotoxicity of anticancer doxorubicin
is significantly reduced (8098%), when the drug is covalently bound to dendrimer via an acid-
labile hydrazone linkage and the drug is successfully taken up by several cancer cell lines. The
encapsulation behaviour of these compounds for the anticancer drugs doxorubicin and methotrexate
has been studied. The highest encapsulation efficiency, with an average 6.5 doxorubicin molecules
and 26 methotrexate molecules per dendrimer, is found for the G = 4 PAMAM terminated with
PEG2000 chains. The anticancer drug 5-fluorouracil encapsulated into G = 4 PAMAM dendrimers
with carboxymethyl PEG5000 surface chains suggests reasonable drug loading, and reduced release
rate and hemolytic toxicity as compared to the non-PEGylated dendrimer [118] as shown in Fig. 7.

Fig 7 The encapsulation of anticancer drugs methotraxate [114].

Dendrimers-ligand Conjugate : Dendrimers can be conjugated with a ligand specific for

targeting tumor sites. Several ligands can be recognized by the appropriate cell surface receptor and
thus internalized where it can deposit the therapeutic agent. One of the ligand such as folate appears
to be an excellent ligand because many cancerous tumor cells overexpress folate receptors. Therefore,
folate conjugated dendrimers can be effective drug deliverers having high affinity for these cancer
cells [119].
206 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

Folate conjugated dendrimers enter the cell via endocytosis. For migration, it must be
hydrophilic and also having a high lipid solubility. Upon entering the cell, the folate conjugates
remain functional for some time. The endocytosed vesicle is then signalled to release the conjugated
moiety into the cytoplasm and the folate receptor returns to the cell surface. 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 [120].
Glyco-dendrimer : This is used as a targeted anticancer drug vehicle because of the relationship
of glycosylation (modification) 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 [121]. Glycodendrimers can be used in a similar manner by being introduced at 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 [122].
Peptide base dendrimer : It consists 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 [123]. 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 [99].
pH sensitive dendrimers : Dendrimers of this type are 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 [116]. 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
[114]. 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 [124].
Dendrimers in Gene Transfection
Dendrimers can act as vectors, in gene therapy. PAMAM dendrimers have been tested as
genetic material carriers. Numerous reports have described 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 [118]. A transfection reagent called SuperFectTM consisting of
activated dendrimers is commercially available. Activated dendrimers can carry a larger amount of
genetic material than viruses. SuperFectDNA complexes are characterized by high stability and
provide more efficient transport of DNA into the nucleus than liposomes. The high transfection
International Journal of Current Chemistry : Vol. 1, No. 4, October-December 2010 207

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 permits the dendrimer to buffer the pH change
in the endosomal compartment (Figure 8) [100]. It should be noted that dendrimers of high structural
flexibility and partially degraded high-generation dendrimers (hyper branched architectures) appear
to be better suited for certain gene delivery operations than intact high-generation symmetrical
dendrimers [121].

Fig 8 Dendrimer in gene delivery (Modified from [99])

6. ISSUES IN DENDRIMERS AS DRUG DELIVERY AGENTS

Dendrimers are particularly attractive as they offer a high drug-loading capacity. The two
methods for the use of dendrimer as drug delivery agents are encapsulation of drugs and dendrimer
drug conjugates.
Non-covalent Encapsulation of Drugs / Host Guest Relation
Encapsulation of drugs uses the satiric bulk of the exterior of the dendrimer or interactions
between the dendrimer and drug to trap the drug inside the dendrimer. Maciejewski introduces the
concept of encapsulating guest molecules into special, egg-shell-like structures [117]. Such a system
can be used to encapsulate drugs and provide controlled delivery. For example, in early studies,
DNA is complexed with PAMAM dendrimers for gene delivery applications, and hydrophobic drugs
and dye molecules are incorporated into various dendrimer cores. An advantage of using dendritic
unimolecular micelles rather than conventional polymeric micelles is that the micellar structure is
maintained at all concentrations because the hydrophobic segments are covalently connected.
Although the introduction of stabilizing poly (ethylene oxide) PEO chains on the dendrimer
periphery has expanded the scope of dendritic unimolecular micelles to incorporate anticancer drugs
208 Hyaluronan-mediated CD44 Receptor Cancer Cells Progression..............

such as 5-fluorouracil methotrexate and doxorubicin, this can slow the drug release rates in these
systems to some extent. A promising new approach to controlling the release of drugs from the
encapsulating micellar compartment involves the use of hybrids of PEO and dendrimers with pH-
sensitive hydrophobic acetyl groups on the dendrimer periphery [117].
Covalent DendrimerDrug Conjugates
An alternative approach to the development of dendrimers as anticancer drug carriers is to
exploit their well-defined multi valency for the covalent attachment of drug molecules to the dendrimer
periphery. In dendrimerdrug conjugates, the drug is attached through a covalent bond either directly
or via a linker/spacer to the surface groups of a dendrimer. Dendrimers have been conjugated to
various biologically active molecules such as drugs, antibodies, sugar moieties and lipids .The drug
loading can be tuned by varying the generation number of the dendrimer, and release of the drug can
be controlled by incorporating degradable linkages between the drug and dendrimer. Conjugates of
PAMAM dendrimers with cisplatin, a potent anticancer drug with non-specific toxicity and poor
water solubility have been delevoped. The conjugates show increased solubility, decreased systemic
toxicity and selective accumulation in solid tumors [114].
7. CONCLUSION AND FUTURE OUTLOOK
HA and ligand CD44 interaction could be a key player in tumor progression in tumor cells.
HA main ligand of CD44 receptor cells, can mediate CD44 cytoskeleton protein activation in tumor
cells leading to cytoskeleton mediated cancer progression. Both membrane bound and cytoplasmic
proteins are involve in signal cascade tumor progression pathway. These cytoskeleton proteins could
be suitable markers for cancer drug delivery. The dynamic features of tumor cell growth, invasion
and metastasis are of great concern to combat deadly cancer or solid tumors. A targeted controlled
drug delivery system is required to curb the tumor cell growth, invasion and metastasis. Dendrimer
has a great potential drug delivery carrier for anticancer or tumor drugs. Highly branched,
monodisperse, multivalent characteristics along with tunable generations make them potential carrier
for targeted drug delivery for treatment of cancer/tumor. Highly soluble nature of dendrimer due to
modifiable charge group and packet globular structure make them of great interest in drug delivery
applications Drug molecule can be easily trapped inside or attached to the periphery without any
leakage or dose concentration problem. Cytotoxity is the main issue for carrier molecule but due to
modifying behaviour of side groups, it can be easily tuned. Not only drug molecule, but ligands and
DNA attached to carrier molecule are also avenues for potential transfection.
8. ACKNOWLEDGMENTS
We wish to thank Universiti Teknologi Petronas for the scholarship awarded to Gul- e-Saba.
We also thank Ms. Anis Suhaila Shuib from the Department of Chemical Engineering, Universiti
Teknologi Petronas and Mr. Mohammad Naveed Zafar from the Department of Chemistry, University
of Auckland, New Zealand for the discussion and some suggestions during the completion of the
manuscript.
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