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Advanced Drug Delivery Reviews 57 (2005) 2215 – 2237
www.elsevier.com/locate/addr
Dendrimer biocompatibility and toxicity B
Ruth Duncan a,*, Lorella Izzo a,b
a
b
Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK
Universita` degli Studi di Salerno, Dipartimento di Chimica, via S. Allende- 84081 Baronissi (SA), Italy
Received 14 April 2005; accepted 13 September 2005
Available online 16 November 2005
Abstract
The field of biomedical dendrimers is still in its infancy, but the explosion of interest in dendrimers and dendronised
polymers as inherently active therapeutic agents, as vectors for targeted delivery of drugs, peptides and oligonucleotides, and as
permeability enhancers able to promote oral and transdermal drug delivery makes it timely to review current knowledge
regarding the toxicology of these dendrimer chemistries (currently under development for biomedical applications). Clinical
experience with polymeric excipients, plasma expanders, and most recently the development of more dclassical polymerTderived therapeutics can be used to guide development of bsafeQ dendritic polymers. Moreover, in future it will only ever be
possible to designate a dendrimer as bsafeQ when related to a specific application. The so far limited clinical experience using
dendrimers make it impossible to designate any particular chemistry intrinsically bsafeQ or btoxicQ. Although there is widespread
concern as to the safety of nano-sized particles, preclinical and clinical experience gained during the development of polymeric
excipients, biomedical polymers and polymer therapeutics shows that judicious development of dendrimer chemistry for each
specific application will ensure development of safe and important materials for biomedical and pharmaceutical use.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Dendrimer; Dendron; Biocompatibility; Toxicity; Polymer therapeutic; Biomedical use
Contents
1.
2.
3.
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Dendritic architectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Biomedical use of dendritic systems — which route of administration? . . .
Dendrimer biocompatibility or toxicology: some general reflections on terminology
Biological properties of dendrimers in vitro . . . . . . . . . . . . . . . . . . . . .
3.1. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Haemolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B
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This review is part of the Advanced Drug Delivery Reviews theme issue on bDendrimers: a Versatile Targeting PlatformQ, Vol. 57/15, 2005.
* Corresponding author. Tel.: +44 29 20874180; fax: +44 29 20874536.
E-mail address: [email protected] (R. Duncan).
0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2005.09.019
2216
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
3.3. Complement activation . . . . . . . . . . . . . . . . . .
3.4. Effect of dendrimers on intestinal tissue and CaCo-2 cell
3.5. Endocytic uptake and intracellular fate . . . . . . . . . .
4. Evaluation of dendrimer properties: in vivo . . . . . . . . . . .
4.1. Biodistribution of dendrimers and dendronised polymers
4.2. General toxicity . . . . . . . . . . . . . . . . . . . . .
4.3. Immunogenicity — a problem or an asset? . . . . . . .
5. Effect of dendrimers on cytokine and chemokine release . . . .
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Background
There is a recognised need to identify novel
therapeutic strategies able to bring improved treatments for life threatening and debilitating diseases [1].
The search for better diagnostics, more selective
medicines, and approaches designed to either engineer
new tissues or promote in situ tissue repair is ongoing.
The technologies proposed are becoming ever more
sophisticated. In this context, the current status of
biomedical applications of dendrimers is reviewed
elsewhere in this volume [2]. More generally, the last
decades have seen research at the interface of polymer
chemistry and the biomedical sciences giving birth to
the first nano-sized (5–100 nm) polymer-based
pharmaceuticals. This family of constructs has been
called bpolymer therapeuticsQ [3,4], even though some
use the bfashionableQ, broader descriptors of nanomedicines, nanospheres, nanocontainers and nanodevices particularly to describe dendrimer-derived
constructs. Whilst some of these terms can be
justified, particularly if a dendrimer is used simply
as a container to entrap a dye, drug or radionuclide,
there is a need to clarify the terminology, particularly
if a dendrimer-based system is destined for clinical
development as regulatory requirements are quite
distinct for novel excipients, therapeutics, formulations and devices. The term bpolymer therapeuticsQ
has been more carefully defined [3,4], and it will be
used here to include those dendrimers proposed as
drugs or multi-component conjugates.
Polymer therapeutics are complex, hybrid, technologies, often combining several components, e.g.,
polymer, drug, peptide, protein, glycoside or oligonucleotide. All use water-soluble synthetic or natural
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monolayer viability.
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2226
2226
2226
2229
2229
2230
2231
2231
2232
2232
polymers to create macromolecular drugs, polymerdrug and polymer-protein conjugates, polymeric
micelles containing covalently bound drug and those
multicomponent polyplexes used for DNA delivery.
This descriptor reflects a regulatory authority perspective which sees these compounds as new chemical entities (NCEs) rather than conventional drug
delivery systems or formulations which simply entrap,
solubilise or control drug release without resorting to
chemical conjugation. Successful clinical application
of polymer-protein conjugates (reviewed in Ref. [5]),
promising clinical results arising from trials with
polymer-anticancer drug conjugates and polymeric
imaging agents (reviewed in Refs. [4,6]), and the
pressing need for non-viral vectors for effective
intracellular delivery of oligonucleotides and proteins
[7] is leading to exponential growth in this field.
Consequently, many different polymer chemistries are
being explored. Transfer of the first dendrimer-based
MRI imaging agent (SH L 643A; Gadomer-17) into
clinical development by Schering [8] and last year,
StarPharma’s initiation of clinical trials with the first
dendrimer-based pharmaceutical, a topically applied
vaginal virucide (Vivagelk) [9] have been important
milestones in this field.
Historically, both successful and failed clinical
trials involving polymer therapeutics (reviewed in
Ref. [4]) have underlined the need for careful choice
of polymer for each intended application. The most
successful early constructs were synthesised using
classical linear, random coil polymers, such as
polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, poly(glutamic
acid) (PGA), poly(ethyleneimine) (PEI) and dextrin
(a-1,4 polyglucose). Although these polymers have
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
generated conjugates [6,10] and polyplexes [11] that
have now been taken into the clinical trial, such
polymers and their conjugates present specific challenges for pharmaceutical development. A manufactured drug substance must be reproducible batch to
batch, be composed of a single, defined species,
whose identity (and impurities) and stability can be
specified using validated techniques, and whose
pharmacokinetics and therapeutic index (activity and
toxicity) can be precisely defined. Many linear
polymers and resultant conjugates are inherently
heterogeneous. Often preparations contain individual
molecules of different chain length (i.e. they are
polydisperse) and heterogeneous structure. This variation in molecular weight can have a profound effect
on biological activity in terms of toxicity and efficacy,
Although it is possible to define acceptable product
specifications for complex polymer therapeutics,
second generation constructs are seeking to minimise
polymer heterogeneity. The advent of dendrimer
chemistry has brought many potential advantages,
synthetic water-soluble macromolecules of defined
chemical composition, tailored surface multi-valency
and of defined 3D architecture [12–15]. However, the
complexity of many synthetic procedures presents
different challenges in terms of cost-effectiveness of
manufacture, challenging molecular characterisation
[16], and the frequently raised question — will
dendrimers be safe for clinical use?
1.1. Dendritic architectures
When discussing dendrimer toxicity it is very
difficult to generalise. A vast array of dendrimer
chemistries and hybrid dendritic architectures are
currently being studied and some are shown schematically in Fig. 1a. Synthesis is conducted using either
synthetic or natural elements (e.g., amino acids,
sugars and nucleotides) as the basic building blocks,
but the dendrimer surface is frequently further
decorated with addition of other bioresponsive elements, recognition moieties, drugs, radioisotopes,
fatty acids, lipids, and sometimes even polymers
(e.g., PEG) (Fig. 1b). Whilst a detailed overview of
dendrimer chemistry is out with the scope of this
article (see Refs. [15,17–19]) the fundamentals are
briefly described here to allow discussion of toxicological implications of the main routes to synthesis.
2217
Dendrimers, also called arborols or cascade molecules, are macromolecules (typically 5000 to 500,000
g/mol) born out of innovative organic chemistry over
thirty years ago [12,13]. Distinguishable from the
hyperbranched polymers that display random branching, dendrimers are well defined, regularly branched
macromolecules. They offer particular advantages
including their nanoscale spherical architecture (at
higher generation), narrow polydispersity and the
multifunctional surface offering the possibility to
tailor-make their surface chemistry. The relatively
empty intramolecular cavity can be amenable to a
host-molecule entrapment providing opportunities for
subsequent controlled drug release. Meijer et al.
proposed the notion of a bdendritic boxQ [20]. The
commercially available polyamidoamine (PAMAM;
Starburstk) dendrimers [13] and poly(propylenemine)
(also called PPI, DAB; AstramolR) dendrimers (Fig.
1c) have been most widely studied for biomedical use.
These dendrimers are prepared by divergent synthesis.
This involves stepwise repeated addition of monomer,
beginning from a multifunctional core, the valency of
which defines the starting number of branching points.
Addition of successive layers (generations) gradually
increases molecular size and amplifies the number of
surface groups present. As PAMAM dendrimers grow
through generations 1–10 their size increases from 1.1–
12.4 nm [21]. These dimensions have been compared
to those of proteins (3–8 nm), linear polymer-drug
conjugates (5–20 nm) and viruses (25–240 nm). So far
PAMAM dendrimers with an –NH2 surface (e.g.,
oligonucleotide delivery), a –COOH surface (e.g.
dendrimer platinates) and an –OH surface (e.g.,
dendrimer-derived magnetic resonance imaging
(MRI) imaging agents) have been most widely studied
for medical applications.
Dendrimers can also be synthesised starting from
the surface using the convergent approach to produce
wedge shaped units or dendrons that can be, as a last
step, joined to the multivalent core [14,22]. This route
has less risk of defects in the final structure (for
example the incomplete branching that can often
occur during divergent synthesis) and it also produces
dendrons that can be used to prepare dendronised
polymers. Throughout the 1990s an ever more
complex array of dendritic architectures has emerged
(Fig. 1). Structures reported include dendritic hybrids,
dendronised polymers, dendrigrafts, cyclodextrin-den-
2218
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
(a)
“simple” dendrimers
dendronised polymers
di-substituted
convergent
divergent
mono-substituted
“complex” hybrids
dendrimer-cyclodextrins
self assembling dendrimer vesicles
dendrimer gels polymers
“dendrisomes”
(b)
drug encapsulation
in the core
surface modification
with targeting ligands
A bioresponsive or
biodegradable core
surface modification
With polymer, oligomer
or fatty acid
surface modification
with additional chemistry
(maybe linker) and
drug attachment
Fig. 1. Diagram showing schematically (a) dendritic architectures under development for biomedical and (b) approaches for design of
therapeutics and drug delivery systems.
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
(c)
O
NH
N
NH2
NH
O
O
O
HN
HN
H
N
HN O
O
N
N
O
O
O
N
N
NH2
H2N
NH
O
N
O
O
N
O HH N
2
NH
HN
HN
NH
NH
NH
O
O
O
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H2N
H2N
N
N
NH2
NH2
NH2
N
N
N
N
N
N
H2N
NH2H N
2
H2N
N
H2N
N
N
NH2 H N
2
N
NH2 NH2
H2N
PAMAM
N
N
NH2
O
N
N
N
N
N
NH2
NH2
NH2
NH2
N
N
N
N
H2N
N
N
DAB
N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
O
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N
N
OH
O
NH2
NH2
NH2
N
N
OH
O
NH2
N
N
H2N
NH2
NH2
NH2
N
N
N
H2N
H2N
N
N
N
H2N
O
NH2 NH2
N
N
H2N
H2N
NH
O
NH2H2N
N
N
H2N
H2N
H2N
NH2
N
N
N
H
N
HN
H2N
N
H2
N
N
H2N
HN
N
O NH
N
H2N
O
NH
H2N
O
H2N
H2N
HN
H
N O
HN
H2N
H2N H2N
H2N
N
O
N
NH
H2N
O
H2N
N
O
NH2O
NH2
NH
HN
HN
H
N
O
NH
O
OH
O
H2N
O OH
NH2
NH2
N
NH2
2219
OH
O
O
O
O
O
O
O
OH
OH
O
O
O
OH
OH
O
O OH
O OH
O
O
O
OH
OH
O
O
O
O
O
O
O
O
OH
OH
OH
OH
O
O
O
OH
PEG-polyester dendron
Fig. 1 (continued ).
drimer hybrids, core-shell architectures, cascade-release dendrimers and self assembling dendrisomes
(see Refs. [23–27]). Of particular interest are the
linear-dendritic hybrids introduced by Fre´chet et al.
[28], and the PEG-poly(ester) dendritic hybrids are
particularly appealing for drug delivery applications
[29,30] (Fig. 1c).
During any discussion on safety it should be
noted that the vast majority of dendrimers so far
synthesised were never intended for pharmaceutical
or biomedical use. The number of studies evaluating
biological properties of dendrimers is growing, but
still more than 75% of the papers published describe
purely synthetic chemistry (new concepts) or nonbiological applications of dendritic systems. Most
dendrimer chemistries reported have poor solubility
in aqueous solutions, and a structure that would
predict likelihood of cellular accumulation, unacceptable toxicity and/or immunogenicity if administered
parenterally. Irrespective of proposed application,
care should be taken during dendrimer manufacture
(particularly on the large scale) to ensure containment. It is well documented that the large surface
area to volume ratio of all nanosized particles can
potentially lead to unfavourable biological responses
if they are inhaled and subsequently absorbed via the
lung [31,32], or swallowed and then absorbed across
the gastrointestinal tract.
1.2. Biomedical use of dendritic systems — which
route of administration?
The majority of studies examining biomedical
properties of dendrimers so far document only in
vitro properties. The proposed clinical applications are
however wide and varied and the most widely
explored biomedical applications are reviewed in
several publications [33–37] and summarised in Table
1. They include development as: inherently active
drugs (e.g., as antiviral and antimicrobial agents and
modulators of angiogenesis); drug-carriers for targeting and controlled release (particularly as anticancer
agents); non-viral vectors to promote oligonucleotide
and gene delivery. Two dendrimer-derived products
are already on the market as in vitro transfection
agents (PolyFectR and SuperfectR). Dendrimers are
also being studied as medical imaging agents, as
components of tissue engineering scaffolds and as
adjuvants for vaccine delivery (reviewed in Refs.
[2,17,33]).
To discuss dendrimer safety there is a need to
consider the likely route and frequency of adminis-
2220
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
Table 1
Proposed biomedical applications of dendrimers and routes of administration
Goal
Examples
Routes of
administration
Refs
Gene and oligonucleotide delivery
PAMAM dendrimers gene delivery
PAMAM and DAB dendrimers oligonucleotide delivery
a-cyclodextrin dendrimers for gene delivery
anionic dendrimers for antisense delivery
Fas ligand delivery
PAMAM-PEG-PAMAM for gene delivery
Dendritic polylysine for transfection
Polyplex mediated delivery
In vitro
In vitro
In vitro
In vivo
In vitro
In vivo
In vivo
In vitro
Pulmonary
i.v.
i.p., i.v., i.t
[38,39]
[40,41]
[42]
Targeting of anticancer chemotherapy,
radiotherapy and BNCT
In vivo diagnostics
As anti-infective agents
Oral delivery
Transdermal delivery
Ocular applications
Vaccine and peptide delivery
Scaffolds for tissue engineering
PAMAM gen. 3.5-Pt
Boron neutron capture therapy (BNCT)
Dendrimer-radioactive gold for targeting tumour
vasculature
125
I-labelled biotinylated dendrimers for antibody
pretargeting
Antitumour activity of glucan derivatives
Folate and antibody targeted dendrimers
Dendrimers for delivery of angiostatin and TIMP-2
PEG-polyester dendron-doxorubicin
N-acetyl-glucosamine-coated glycodendrimers as
antitumour agents
Gadomer MRI contrast agents for vascular imaging
PAMAM and DAB dendrimer-MRI agents for functional
kidney or liver imaging
MRI imaging agents for tumour blood vessels
MRI imaging agents based on PEG-dendrimers
Vivagel
Anti-HIV fifth generation DAB dendrimer (PS Gal
64mer),functionalized with 3-(galactosylthio)propionic
acid residues
Sulfated galactose modified dendrimers
Dendrimers to entrap and solubilise agents
PAMAM-propanolol to bypass pgp efflux
Indomethicin delivery using PAMAM gen 4, gen 4–OH
or gen 4.5
Antiangiogenic activity of a peptide-dendrimer
delivering natisense
PAMAM-glucose and glucose-6-sulfate to reduce
antigenesis and scar formation
Peptide vaccine delivery, TAT and Fos peptide delivery
tration in the proposed applications. Pharmacokinetics, biodistribution and fate are key issues that,
together with the nature of the disease to be treated
(acute or chronic, life-threatening), ultimately define
whether the risk-benefit (in terms of potential safety/
efficacy) of any novel technology can be justified.
The MRI imaging agent SH L 643 (Gadomer-17)
undergoing clinical evaluation [8] is administered
[43]
[44]
[45]
[46]
[47]
[48]
[49–51]
[52]
[53]
In vivo
i.v.
Topical
In vitro
[54]
[55–57]
[58]
[29,30]
[59]
[8]
[60–63]
[64–66]
[67]
[9]
[68]
Topical
[69]
[70–73]
[74]
[75,76]
In vitro
[77]
Local in the eye
[78]
Oral
[79–81]
[82]
intravenously (i.v.). In this case the need for repeated
administration is unlikely. Administration i.v. is also
typically used for parenteral targeted delivery applications, e.g., tumour targeting. In this case repeated
administration during a course of treatment is usually
required. Some experimental studies have used intratumoural (i.t.) injection of dendrimer-based oligonucleotide delivery systems, but this route is rarely used
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
in clinical practice, as it is not an option for targeting
disseminated, metastatic disease.
The virucide (Vivagelk) [9] is applied topically in
the vagina where it is expected to act locally. An
increasing number of studies are proposing dendrimers
as topical enhancers for transdermal delivery, and also
as transport enhancers in other situations, e.g., for oral
delivery and delivery of drugs across the blood brain
barrier. Pulmonary delivery has also been suggested as
a route of administration for gene delivery.
2. Dendrimer biocompatibility or toxicology: some
general reflections on terminology
Should we refer to dendrimer bbiocompatibilityQ or
btoxicityQ? The pharmaceutical industry refers to a
drug in terms of its btoxicityQ. This is a measure of
non-specific, unwanted harm it may elicit towards
cells, organs, or indeed the patient as a multi-organ
system (Fig. 2). The field of biomedical materials has
over recent decades seen the development of sophisticated, novel polymeric biomaterials, for example,
hydrogels used as soft contact lenses, protheses
(including stents) and devices. These biomedical
In vitro testing
• Cytotoxicity (panel of cell
lines, 72 h)
• Haematocompatibility
- RBC lysis (Hb release)
- Complement activation
• Ability to induce cytokine release
• Biodegradation of the polymer
- cytotoxicity of degradation products
In vivo testing
• Body distribution
- short term fate (1 h)
- long term fate (1 month)
• Definition of organ specific
toxicity e.g. liver, kidney
• Intracellular fate
- endocytic pathways
- fate of the polymer, degradation
• †Pharmacological activity of the
construct
• Immunogenicity
- IgG and IgM induction
- cytokine induction
• Metabolic fate
Preclinical testing
• Teratogenicity
• Therapeutic index
• Single dose and multiple dose toxicity
• Metabolic fate
Patient
Fig. 2. Methods used to assess polymer toxicity in vitro and in vivo.
2221
materials increasingly combine physical function with
a local drug delivery application, and the term
bbiocompatibilityQ is commonly adopted by convention to describe their biological properties.
bBiocompatibilityQ was defined at a consensus conference of the European Society for Biomaterials in
1986 as dthe ability of a material to perform with an
appropriate host response din a specific applicationT
[83]. This useful definition highlights the need to
consider the suitability of a material both in respect of
its potential detrimental effect in the body (toxicity),
and in respect of the potential detrimental or beneficial
effect of the physiological environment on the
performance of the material (Fig. 2). This definition
also stresses the need to define a material
bbiocompatibilityQ only in the precise context of its
use. If a dendrimer or dendron is used as a component
of a biomedical material, e.g., a tissue engineering
application the biocompatibility of the material must
be defined. It should be emphasised that it is never
possible to define any dendrimer (polymer, material)
chemistry as biocompatible or non-toxic without
qualification as to the intended precise use.
Although concerns have been expressed as to the
potential hazards of novel polymeric materials, it
should be stressed that the development of dendrimers
as polymer therapeutics requires no more, or less,
caution than observed for any other drug or formulation. When developed as covalently linked drug
carriers, the dendrimer and drug are viewed as the two
primary metabolites after parenteral administration
and the subsequent fate of each will require documentation. The safety issues considered for HPMA
copolymer anticancer conjugates, the first synthetic
conjugate to enter clinical trial, are well documented
and a good guide for emerging dendrimer-based
therapeutics. The immunological and haemocompatibility properties of HPMA copolymers [84–90] were
established early in their history, as was the effect of
HPMA copolymers of Mw (10,000–800,000 g/mol)
on biodistribution after i.v. administration [91]. This
information was used to define a molecular weight
range amenable to renal elimination and thus safe for
clinical use. In addition, the potential danger of
cellular accumulation of non-biodegradable polymers
(leading to lysosomal storage disease) is well known
[92]. It is important to define whether any new
polymer (or dendrimer) proposed for parenteral use
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R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
will be biodegradable, and over what time frame
degradation will take place. The importance of
rational design of new polymer therapeutics in respect
of their proposed use cannot be over emphasized.
Once a clinical candidate is identified it is essential
to define the properties of the compound per se not
just the component drug and carrier molecule. For
example, to justify entry of each HPMA copolymer–
anticancer conjugate in to clinical trial [93], it was
necessary to conduct a series of compound-specific
preclinical toxicity tests. Most of this information is
not in the public domain, but the bGood Laboratory
PracticeQ (GLP) single and chronic dose study
involving PK1 (FCE28068) used to select the starting
dose for its Phase I clinical trial has been reported
[94]. Demonstration of reduced cardiotoxicity of PK1
was also a preclinical requirement [95].
As a general rule, for any polymeric carrier to be
suitable for parenteral application it is essential that the
carrier is non-toxic and non-immunogenic, and it
should preferably be biodegradable. It must display
an inherent body distribution that will allow appropriate tissue targeting — to the desired site, but away from
sites of toxicity. During the 1980s we began to develop
a series of in vitro and in vivo tests (Fig. 3) and these
have been routinely used since as a pre-bscreenQ of new
polymers (including dendrimers) under consideration
as potential drug delivery systems. Simple in vitro
assays such as cytotoxicity against a panel of cell lines
(72 h; MTT assay) [96] and red blood cell haemolysis
assays (1 h, 5 h and 24 h) [97] were established to assess
Biocompatibility
Biological
Environment
adverse or
beneficial
effect on
material
performance
Material
or Device
adverse
effect
Biological
Environment
whether preliminary in vivo evaluation is ethically
justifiable, since these tests have been adopted by
many. Usually dextran (Mw ~70,000 g/mol) is used as
a negative control and polyethyleneimine (PEI) or
poly-l-lysine (PLL) as positive reference controls for
these studies. To monitor more subtle changes of cell
morphology or cell aggregation during incubation with
polymers scanning electron microscopy (SEM) has
been a useful tool [97]. As toxicity of a polymer in vivo
is profoundly influenced by its pharmacokinetics and
biodistribution, biodistribution studies form an important part of the early polymer screening. This helps to
identify opportunities or barriers to cell specific
targeting in vivo before lengthy, but irrelevant in vitro
tests are undertaken. It also helps to identify those
organs/tissues exhibiting maximum deposition and
thus likely toxicity-target organs. Features such as
polymer molecular weight, charge and hydrophobicity
and more subtle aspects of physico-chemical solution
behaviour can have a profound effect on biodistribution
particularly if a polymer is carrying a surface disposed
drug payload.
Any early screen must examine the antigenticity
(IgG, IgM production), and cellular immunogenicity
(cytokine and chemokine induction) is essential for
any novel polymer/dendrimer destined for parenteral
use. Repeated parenteral administration of macromolecular drugs (or conjugates), including proteins, is
well known to induce antibody formation if the
construct is seen as bforeignQ. This can lead to
anaphylactic shock. In addition, a non-immunogenic
carrier is likely to produce a hapten effect when it
presents surface moieties such as the drug, peptide, or
saccharide. Inherent immunogenicity can preclude
future use for many applications.
3. Biological properties of dendrimers in vitro
Toxicology
3.1. Cytotoxicity
Material
Or Drug
adverse
effect
Biological
Environment
Fig. 3. Difference between bbiocompatibilityQ and btoxicityQ.
Many studies have examined dendrimer cytotoxicity in vitro. They use different cell lines, a variety of
incubation times (hours to days), and various assay
methods (for examples see Refs. [98–107]). The
maximum concentration of dendrimer used is frequently relatively low, and time of cell exposure short — the
duration of exposure chosen to match the contact time
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
during a transfection or pharmacological assay. Variability in cell culture conditions normally makes it
difficult to directly compare experiments conducted in
different laboratories. To define cytotoxicity of a new
dendrimer chemistry it is most appropriate to use a
physiologically meaningful incubation time (exposure
could be days, weeks or months if the dendrimer is to
be administered parenterally) and high enough concentrations to define the inhibitory concentration
diminishing viability by 50% (IC50 value).
Nevertheless, some general trends are clear. As has
been widely documented for other polycations, dendrimers bearing –NH2 termini display concentrationand usually generation-dependent cytotoxicity. When
incubated with V79 Chinese hamster lung fibroblasts
(24 h) Roberts et al. [99] showed that cationic PAMAM
dendrimers caused a decrease in cell viability. The
concentration producing 90% cell death was 1 nM (~7
ng/ml) for generation 3, 10 AM (~280 Ag/ml) for
generation 5, and 100 nM for generation 7. We
obtained similar results when comparing the cytotoxicity of PAMAM, DAB, and diaminoethane (DAE)
dendrimers using poly(l-lysine) (PLL), polyethyleneimine (PEI) and dextran as reference materials [100].
We used three cell lines (B16F10 murine melanoma,
CCRF human lymphoblastic leukaemia and HepG2
human hepatoma) and a 72 h incubation time with the
MTT assay to assess viability. Cationic PAMAM
dendrimers generations 1–4 were cytotoxic and displayed IC50 values, similar to PLL. For example,
generation 4 PAMAM, DAB and DAE dendrimers had
IC50 values = 50–300 Ag/ml. Compared to DAB
dendrimers of equivalent surface functionality,
PAMAM dendrimers were slightly less toxic [100].
B16F10 morphology visualised by scanning electron microscopy (SEM), changed dramatically on
exposure to DAB and DAE dendrimers (1 mg/ml) for
1 h (Fig. 4) [100]. Although B16F10 cells incubated
with PAMAM generation 4 (5 Ag/ml) for 1 h showed no
change in morphology, a longer (5 h) exposure at this
low concentration led to appearance of distinctive
craters indicating the beginning of membrane damage.
Such observations are typical of polycation-induced
membrane damage. Interestingly, when Kissel and
colleagues [102,103] investigated the cytotoxicity of a
series of polycations in L929 mouse fibroblasts
(monitoring lactate dehydrogenase (LDH) release and
assessing viability using the MTT assay over 24 h) they
2223
reported minimal toxicity of PAMAM generation 3.
However, it is noteworthy that in these experiments the
polymer solutions were readjusting back to physiological levels to eliminate any polymer-induced changes in
medium osmolarity and pH. One could argue that such
manipulation is not relevant to the in vivo situation
where it is not possible to eliminate pH and osmotic
effects. These authors ranked the order of polymer
cytotoxicity as PEI = PLL N dextran N PAMAM generation 3, and also reported a good correlation between
LDH release, their MTT measure of cell viability and
changes in cell morphology visualised by light
microscopy.
Recent studies have investigated the mechanism of
cationic PAMAM induced membrane damage using
1,2-dimyristoyl-sn -glycero-3-phosphocholine
(DMPC) lipid bilayers and KB and Rat2 cells in culture
[108]. Techniques such as atomic force microscopy
(AFM), and fluorescence microscopy were used to
visualise damage, and release of the enzyme LDH to
explore leakiness of cell membranes. Whilst generation
7 PAMAM dendrimers (10–100 nM) caused the
formation of holes (15–40 nm in diameter) in DMPC
lipid bilayers, generation 5 dendrimers were only able
to expand holes at existing defects. Using a BCA
protein assay it was suggested that generation 5
PAMAM dendrimers were not cytotoxic (up to a 500
nM), however this dendrimer did induce dose-dependent LDH release. Two hours following removal of the
dendrimer it was shown that membrane integrity was
re-established. An acetamide-terminated generation 5
PAMAM did not however affect membrane integrity
(up to a concentration of 500 nM) showing the
importance of –NH2 functionality [108].
Dendrimer cytotoxicity is dependent on the chemistry of the core, but is most strongly influenced by the
nature of the dendrimer surface. For example, the
cytotoxicity (clone 9 cells; 3 h, MTT assay) of the
cationic dendrimers selected from a melamine-based
dendrimer library including amine, guanidine, carboxylate, sulfonate, phosphonate, or PEGylated surfaces,
showed that cationic dendrimers were much more
cytotoxic than anionic or PEGylated dendrimers [104].
Similarly, quaternised PAMAM–OH derivatives
showed a lower level of cytotoxicity than PAMAM–
NH2 because of shielding of the internal cationic
charges by surface hydroxyl groups [109]. Modification of the PAMAM generation 4 surface with lysine or
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R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
Fig. 4. Scanning electron microscopy of B16F10 cells exposed to dendrimers for 1 h at different concentrations, from reference [100].
arginine led to increased toxicity compared to native
PAMAM when incubated with HEP G2 cells or 293
human embryonic kidney cells (48 h; MTT) [101]. This
was attributed to increased charge density and molecular weight. We found that anionic PAMAM generation
1.5–9.5 dendrimers and DAB dendrimers, with a –
COOH surface were not cytotoxic towards B16F10,
CCRF or HepG2 cells up to concentrations of 5 mg/ml
(72 h; MTT assay). SEM of the B16F10 cells exposed
to such anionic dendrimers revealed no morphological
changes [100]. A potentially toxic dendrimer core is
more accessible when presented to cells as a low
generation species due to the more open molecular
structure. In addition, low generation species have
more accessible surface terminal groups, and these
become sterically hindered due to crowding at higher
generations. Increased branching (generation) and a
greater surface coverage with biocompatible terminal
groups like PEG are being widely used to create less
toxic dendrimers. We found that PEO-modified carbosilane dendrimers (CSi-PEO) were generally not toxic
when incubated (up to 2 mg/ml) with CCRF and Hep
G2 cells, although the lowest generation CSi-PEO
dendrimer was surprisingly cytotoxic towards B16F10
cells at higher concentrations [100]. In such cytotoxicity studies variation in cytotoxicity in respect of cell
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
type is commonplace so care should be taken not to
generalise.
Using the same assays described above to test the
PAMAM and DAB dendrimer libraries, we found that
PEG-poly(ester) dendritic hybrids were non-toxic towards B16F10 cells (IC50 N 1 mg/ml; 72 h; MTT) [110].
Indeed Frechet et al. [29] reported that a three-arm PEG
star (Mw = 22,550 g/mol) bearing generation 2 poly(ester) dendrons (final Mw = 23,500 g/mol) had an IC50 of
40 mg/ml towards B16F10 cells during a 48 h incubation. These preliminary observations suggest that this
family of dendronised polymers may have real potential
for further development in drug delivery applications.
3.2. Haemolytic activity
Red blood cell (RBC) lysis is a simple method
widely used to study polymer-membrane interaction. It
gives a quantitative measure of haemoglobin (Hb)
release [96,97]. The data obtained in such assays also
give a qualitative indication of potential damage to
2225
RBC’s of dendrimers administered. Subtle differences
in haemolytic behaviour have been seen according to
the precise structure of the dendrimer under study.
Cationic PAMAM, DAB and DAE dendrimers (except
PAMAM generation 1) demonstrate after 1h generation-dependent haemolysis above a concentration of 1
mg/ml [100]. Unlike PAMAM dendrimers, the DAB
and DAE dendrimers showed no generation-dependency in haemolytic activity [100], and the PEGpoly(ester) dendritic hybrids were not haemolytic
[110]. It should be noted that Fischer et al. [103] failed
to demonstrate any significant haemolysis of PAMAM
generation 3 (up to 10 mg/ml) after 1 h incubation
(b 7% haemolysis), but again this could have been due
to manipulation of incubation pH and osmolarity after
polymer addition in these in vitro studies.
Even at a non-haemolytic concentration of 10 Ag/
ml, cationic PAMAM and DAB dendrimers caused
substantial changes in RBC morphology after 1
h (Fig. 5). RBCs typically adopted a rounded
appearance after incubation with cationic dendrimers
Fig. 5. Scanning electron microscopy of RBC incubated exposed to dendrimers for 1 h, from Ref. [100].
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R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
and cells were often seen to aggregate, probably due
to dendrimer crosslinking. Exposure to higher dendrimer concentrations (1 mg/ml) exaggerated this
dclumpingT behaviour and membrane damage was
evident. Surprisingly Chen et al. [104] found that
melamine-derived cationic dendrimers were more
haemolytic (at both 1 and 24 h) than anionic or
PEGylated melamine dendrimers. These observations
were consistent with the lack of haemolytic activity
seen for anionic PAMAM and DAB dendrimers, and
also the PEO modified CSi dendrimers (up to
concentration of 2 mg/ml; 1 h) [100]. RBCs exposed
to the anionic PAMAM dendrimers of generation 3.5
to 9.5 showed no morphological changes (up to a
concentration of 2 mg/ml) [110]. Preliminary studies
with polyether dendrimers found that compounds
with carboxylate and malonate surfaces were not
haemolytic at 1 h, but, unlike anionic PAMAM
dendrimers, they were lytic after 24 h [100].
3.3. Complement activation
PAMAM dendrimers and high molecular weight
PLL and PEI are all strong complement activators
[111]. Activation by PAMAM dendrimers was equivalent to that caused by PLL, but less than that seen with
the branched PEI polymer. It has also been shown that
generation 5 PAMAM dendriplexes (charge ratios
N1 : 1) activate the complement system after 1 h incubation at 37 8C due to a net positive charge. Although
haemolytic activity of such complexes is markedly
decreased, heamagglutination can be seen following
incubation of RBCs with dendriplexes after several
hours [112]. Although dendriplex formulations may be
more suitable for i.v. injection than cationic dendrimers
alone, it is important to note that dissociation of the
complex, either in transit or at the target site, will
liberate the individual components, which may then
themselves locally exhibit biological activity so care is
warranted.
3.4. Effect of dendrimers on intestinal tissue and
CaCo-2 cell or CaCo-2 monolayer viability
With increasing interest in the possibility of using
dendrimers for oral delivery, toxicity has also been
examined using a variety of gastrointestinal tract
models. We used the rate of active transport of
glucose across the rat everted gut sac in vitro as a
measure of PAMAM dendrimer toxicity. Generations
2.5 and 3.5 and generation 4 at concentrations up to
100 Ag/ml (over 2 h) were studied [113]. In general
tissue exposed to anionic PAMAMs showed greater
ability to transport glucose. PAMAM generation 4
caused toxicity in a concentration-dependant fashion
and barrier breakdown at 100 Ag/ml. Using CaCo-2
cells as an in vitro model combined with the MTT
assay as a measure of cell viability, Jevprasesphant
et al. [105,106] systematically studied the cytotoxicity of cationic PAMAMs (generations 2–4; incubation time 3 h; concentrations up to 100 AM).
Whereas cationic PAMAMs showed concentrationand generation-dependent cytoxicity, the anionic
generations 2.5 and 3.5 were non-toxic. Surface
modification of cationic PAMAM dendrimers by
addition of six lauroyl or four PEG2000 chains
produced a marked decrease in their cytotoxicity
[106].
The effect of PAMAM dendrimers on the integrity
of CaCo-2 monolayers was assessed by measuring
either the rate of transport of [14C]mannitol or changes
in transepithelial resistance (TEER) [105,106]. Whilst
anionic PAMAMs had no effect on TEER, cationic
PAMAM dendrimers reduced the TEER markedly.
For example, generation 2 reduced the TEER values
to 60% of the starting value at a concentration of 10
AM, and moreover to 20% at a concentration of 700
AM. Although transepithelial resistance did recover
over time once dendrimer is removed, it is an
interesting debate as to whether the initial disruption
of a functional barrier would be a useful tool to
promote dendrimer drug conjugate transport across
the epithelial barrier, or simply be harmful in vivo.
3.5. Endocytic uptake and intracellular fate
Few studies have examined the mechanisms of
endocytosis of dendritic polymers in any depth, but it
is clear that their cellular pharmacokinetics will play a
major part in determining biological properties,
including toxicity. Both the rate of internalisation by
particular cell types and the intracellular fate are
important. Mechanism and rate of uptake impacts on a
dendrimers suitability for use in drug, gene or protein
delivery. Subsequent intracellular fate can influence
antigenicity and toxicity. Slowly degrading, or non-
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
biodegradable dendrimers administered repeatedly via
the parenteral route will potentially accumulate in
lysosomes and thus have the potential to create a
lysosomal storage disease syndrome depending on
their frequency and dose of administration [114].
Dendrimers, like other macromolecules, are transported into and across cells via the endocytic
pathways. Experiments conducted in intestinal models suggest that dendrimers can be transcytosed
across the epithelial tissue [113,115]. Fluorescent
and colloidal gold-labelled PAMAM dendrimers
2227
have been visualised inside CaCo-2 cells [115]. We
found that 125I-labelled anionic PAMAMs of lower
generation (1.5 and 2.5) were transported across
everted rat intestinal tissue much faster than other
linear polymers such as polyvinylpyrrolidone (PVP),
poly(N -vinylpyrollidone-co -maleic anhydride)
(NVPMA) copolymers and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers [113]. The latter
display serosal transfer endocytic index (EI) values
in the range 0.1–0.3 Al/mg protein/h (Fig. 6a) In
contrast, the anionic PAMAMs had extremely high
(a)
15
/h))
Endocytic index ( µl / mg protein /h
Tissue
Serosal
10
5
0
PVP HPMA
BSA
NVP
MA
NVP
MA
(2(2-)
NVP
MA
(2+)
Tomato
lectin
G3
G4
G 2.5
G 3.5
G 5.5
PAMAM Dendrime rs
(b)
PAMAM G4
5 h; 0.002 mg/ml
Fig. 6. Cellular transport of PAMAM dendrimers. Panel (a) shows rates of transport of PAMAM dendrimers and other polymers across everted
rat intestinal tissue in vitro (key; polyvinylpyrolidone (PVP), N-(2-hydroxypropylmethacrylamide copolymer (HPMA), bovine serum albumin
(BSA), modified vinylpyrolidone-maleic anhydride copolymers (NVPMA) and tomato lectin. Taken from Ref. [113]. Panel (b) uptake of
PAMAM generation 4 labelled with Oregon Green by B16 F10 cells at 5 h. From Ref. [146].
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R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
serosal transfer EI values in the range 3.4–4.4 Al/mg
protein/h. Only 15–20% of the anionic PAMAM
dendrimer-associated radioactivity remained associated with the tissue, 80–85% being transferred directly
across to the serosal fluid. Transfer of generations 2.5
and 3.5 increased linearly with substrate concentration indicating capture by fluid-phase endocytosis. In
contrast, cationic PAMAM dendrimers showed high
tissue uptake with 55–60% of the radioactivity
associated with generations 3 and 4 located in the
tissue and only 35–40% transferred into the serosal
fluid.
El-Sayed et al. [116] found that transport of
generation 2 PAMAM dendrimers across CaCo-2 cell
monolayers progressed by an energy-dependent process and they suggested that transport occurred by a
combination of paracellular transport and an adsorptive
endocytic mechanism. Addition of generation 2
PAMAM dendrimers did not alter [14C]paclitaxel
transport so it was suggested that a P-gp efflux system
was not affected by these dendrimers. However,
generation 2 PAMAM did cause an increase in
[14C]mannitol permeability consistent with the observed decreased TEER [116].
Increasingly, fluorescent-labelled PAMAM dendrimers incubated with cultured cells have used to
visualise uptake and localisation within intracellular
vesicular compartments [110,116–119] (Fig. 6b)
confirming the potential for their endocytic internalisation. However, interpretation of fluorescence microscopy is fraught with difficulties, and artefacts are
particularly common in fixed cells and/or when using
fluorophores that display pH- and/or concentrationdependant quenching. In addition, it has been shown
that conjugation of hydrophobic fluorophores can
markedly influence cellular uptake mechanisms
[119].
Few have systematically studied the precise
mechanisms of dendrimer internalisation and subsequent fate. Our studies on the endocytic uptake
by endothelial-like ECV304 cells of Oregon green
labelled-linear and star-shaped PEGs bearing generation 1–4 poly(ester)dendrons found that the rate
of uptake and fate varied according to conjugate
architecture [110]. Internalisation was inhibited by
incubation at 4 8C suggesting an energy dependent
process. The higher molecular weight and more
branched the PEG-dendrons the lower the intra-
cellular accumulation and this was found to be
due to an increased rate of exocytosis of these
more branched structures. These findings of
architecture-related properties have been supported
by fluorescence microscopy [110]. Preliminary
studies examining the uptake of Oregon green
labelled-PAMAM generation 4, branched PEI and
linear PEI by B16F10 cells showed that in this
cell line cell association occurred in the rank order
dendritic N branched PEI N linear. Unlike these cationic polymers which show significant membrane
binding at 4 8C [117] the PEGs poly(ester)dendrons appeared to be internalised by fluid-phase
endocytosis.
Superfect-derived dendriplexes incubated with
EA.hy 926 human endothelial cell lines showed
strong binding of the dendriplexes to the plasma
membrane at 4 8C [118]. Further, observation that
transfection was reduced by cholesterol depletion
following addition of methyl-h-cyclodextrin
(MhCD), and was restored by cholesterol replacement led to the conclusion that dendriplexes are at
least in part internalised by a cholesterol-dependant
pathway. FITC-labelled dendriplexes were seen to
co-localise with the ganglioside GM1 present in
membrane rafts, and also the raft marker ChTxB,
observations that also suggest receptor-mediated
uptake via this pathway [118]. Preliminary studies
on the internalisation of Oregon green-labelled
PAMAM generation 4 and Oregon green-labelled
branched PEI by B16F10 cells also suggested
internalisation predominately via a cholesterol-dependent pathway [117].
Recent studies exploring PPI (DAB) dendrimers
for the delivery of a 32P-labeled triplex-forming
oligonucleotide (ODN) showed that dendrimers could
enhance uptake by MDA-MB-231 breast cancer cells
~14-fold [120]. Enhancement of uptake was related
to concentration and dendrimer generation with the
generation 4 showing the most pronounced enhancement of uptake. In this case the hydrodynamic radius
of the PPI dendrimer — ODN nanoparticles formed
was ~130 nm, so it is important to underline the
differences in endocytic processing that will result
from cell exposure to dendrimer alone, dendrimers
carrying a drug payload and the large nanoparticlesized dendriplexes formed following gene or ODN
complexation.
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
4. Evaluation of dendrimer properties: in vivo
4.1. Biodistribution of dendrimers and dendronised
polymers
Biodistribution of parenterally administered dendrimers has been widely studied, particularly in
relation to their development of dendrimer-based
imaging agents designed to monitor the cardiovascular system, liver or kidney function and for imaging
tumour vasculature, their use for tumour targeted
boron neutron capture therapy (BNCT) and as
parenterally administered drug delivery systems. It is
worthwhile to consider their biodistribution in the
context of toxicokinetics. A number of analytical
approaches have been used. These include use of
[14C] [99] and 125I-radio-labelling [100,121], gadolinium (Gd)-labelling for MRI imaging [8] and
analysis of the dendrimer-drug payload distribution
(e.g., platinum by atomic absorption spectroscopy)
[48]. Each of these methods is however an indirect
indication of fate as the marker can become dissociated from the dendrimer. It is important to note that
dendrimer surface modification with a radiolabelled
ligand, gadolinium (via its chelate) or a drug can itself
markedly alter the subsequent biodistribution of the
dendritic carrier per se. In particular, if the surface is
hydrophobised reticuloendothelial capture may predominate. Comparing distribution patterns seen using
different techniques brings some common conclusions. Smaller generation dendrimers are subjected to
rapid renal elimination. Those with charged (cationic
or anionic) or hydrophobic surfaces are rapidly
cleared from the circulation, particularly by the liver.
Dendrimers with a hydrophilic surface (e.g., –OH
termini or PEGylated dendrimers) escape rapid
clearance.
After i.v. administration of 125I-labelled cationic
(generation 3 and 4) and 125I-labelled anionic (generations 2.5, 3.5 and 5.5) PAMAM dendrimers to
Wistar rats we found that cationic dendrimers were
rapidly cleared from the circulation (b 2% recovered
dose in blood at 1 h) [100]. Anionic dendrimers
showed longer circulation times (~20–40% recovered
dose in blood at 1 h) and all displayed generationdependent clearance rates. Both anionic and cationic
species were captured by the liver, and 30–90% of
recovered dose was found there at 1 h [100]. When
2229
the same 125I-labelled PAMAM dendrimers were
injected i.p., radioactivity was transferred into the
bloodstream within an hour and subsequent fate
mirrored that seen following i.v. injection. Wilbur et
al. [121] also showed that 125I-labelled iodobenzoate
(PIB) — biotinylated-PAMAM dendrimers (generations 0,1, 2, 3 and 4) were cleared quickly from the
bloodstream after i.v. administration. In these experiments the highest concentration of radioactivity was
found in the kidney (8–48% dose/g) after 4 h. The
biodistribution of 125I-labelled PEG-poly(ester) dendritic hybrids of molecular weight 3790, 11,500 and
23,500 g/mol was studied after i.v. injection into mice
[30]. It was found that the two smaller compounds
were rapidly eliminated via the kidney whilst the
higher molecular weight compound showed liver
uptake (53% injected dose at 5 h). This was attributed
to the more exposed iodinated-phenol hydrophobic
core of this molecule, but the longer plasma circulation time of this larger dendrimer may also have
played a role and might be pursued to guide further
studies to investigate any organ-specific patho-physiological changes.
Dendrimers are considered particularly attractive as
MRI imaging agents [8,60–67]. As macromolecules
they bring potential for longer blood circulation times
than smaller chelates such as Gd-[DTPA] and
moreover the multifunctional dendrimer surface
brings enhanced relaxivities. Clinical studies in
volunteers with the blood pool contrast agent
Gadomer17 (also called SH L 643A), using an
inversion-recovery 3D image acquisition technique
demonstrated the clinical potential of this technique
with improved MRI imaging of the coronary arteries
[8]. Numerous biodistribution studies with dendrimerbased contrast agents have now been reported. Lower
generation PAMAM-based contrast agents show rapid
blood clearance and renal elimination. However, only
20% of injected dose of a generation 4-based
PAMAM-Gd contrast agent was excreted from the
body during the first 2 days. It was also shown to
transiently accumulate in renal tubules, thus bringing
the potential risk of toxicity if an unstable Gd(III)
chelation released toxic Gd(III) ions [60,61,122–124].
Comparing PAMAM and DAB imaging agents [125],
a significantly larger amount of DAB-Gd was found
to accumulate in the liver compared to the same
generation PAMAM-Gd. Despite this liver accumula-
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R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
tion, the DAB dendrimer-Gd, it was excreted from the
body faster than PAMAM-Gd of the same generation.
However, it should be noted that even the small
generation 2 DAB-Gd imaging agent was retained in
the body (8% of the injected dose 48 h post injection)
much more than Gd-DTPA (N 2% at 48 h). Using MRI
to follow generation 3 PAMAM dendrimer-gadolinium chelates in rats, Margerum et al. [67] also noticed
high liver levels still present at 7 days (1–40%).
PEGylation of the dendrimer surface significantly
increased blood half-life and liver accumulation fell to
1–8% at 7 days. These observations all have
toxicological implications.
There is increasing interest in the use of dendrimers
as vectors for tumour drug targeting. Targeting via
receptor-mediated localisation has largely been studied in vitro and a growing number of targeting ligands
have been explored; e.g., folate [55,56], epidermal
growth factor (EGF) [51], monoclonal antibodies
including cetuximab [49,50,57]. The concept is
appealing and evidence of targeting is easy to
establish in vitro. However, for dendrimers as for
other targeted drug delivery systems, it has proved
more difficult to realise receptor-mediated targeting in
vivo. For example, a boronated generation 4 PAMAM
dendrimer attached to a monoclonal antibody against
B16 melanoma cells (IB16-6) produced immunoconjugates containing very high numbers of boron atoms/
antibody (2200–5000), but studies with both boronfree and boronated PAMAM dendrimers showed
generation-dependent accumulation in the liver and
spleen for both after i.p. injection. The generation 4
PAMAM dendrimer showed 5-fold greater hepatic
uptake than generation 0 dendrimer over 72 h [49].
When 131I-labelled boronated-PAMAM generation
4 containing EGF was injected i.v. into Fischer rats
bearing a C6-EGF transfected glioma relatively low
levels of tumour localisation resulted (0.01% and
0.006% dose/g at 24 and 48 h, respectively) [126].
Non-specific liver and spleen localisation was much
higher (5–12% dose/g). After intratumoural (i.t.)
administration, accumulation of this dendrimer in
the liver and spleen was significantly reduced (0.1%
of injected dose/g), and accumulation in epidermal
growth factor receptor-positive gliomas after 48 h was
significantly higher after i.t. injection (16.3% of
injected dose/g) than after i.v. injection (1.3%) [51].
Intratumoural administration is not however a practi-
cal treatment for life-threatening disseminated metastatic disease.
Passive tumour localisation by the enhanced
permeability and retention (EPR) effect [127] arising
from hyperpermeable tumour vasculature has been
widely used (in vivo and clinically) to target
macromolecular anticancer agents to angiogenic solid
tumours (reviewed in Ref. [6]). In this context we
found that i.v. injection a PAMAM generation 3.5platinate was able to selectively target platinum to s.c.
B16F10 tumours leading to a ~50 fold increase
compared to that seen after i.v. administration of
cisplatin at its maximum tolerated dose (MTD) [48].
Interestingly surface platination of generation 3.5
PAMAM dendrimers led to lower levels of liver
localisation (platinum levels measured by AAS) than
for the parent 125I-labelled dendrimer [100]. Fluorescent [128] and Gd-labelled PAMAM dendrimers
(generation 4–8) have recently been explored as
nanoprobes for monitoring tumour vascular permeability [66], but detailed discussion is beyond the
scope of this review.
4.2. General toxicity
Few toxicological studies involving dendrimer
administration in vivo have been reported. Certainly
PAMAM dendrimers bearing a carboxylate surface
are less toxic than the cationic derivatives. In studies
with cationic PAMAM dendrimers, Roberts et al. [99]
administered generations 3, 5 and 7 to mice at doses
of 2.6, 10 and 45 mg/kg, respectively. The dendrimers
were given either as single dose or repeatedly once a
week for 10 weeks and observation continued for
either a 7 day or a 6 month period. Although no
behavioural changes or weight loss was reported over
a 2 h period, after administration of generation 7 three
animals died. In the multiple dose study a degree of
liver cell vacuolation was also observed during
histopathology and this would be consistent with a
lysosomal storage problem. Further studies are needed
to verify these findings.
We found that three daily doses of PAMAM
generation 3.5 administered i.p. to mice at a daily dose
of 95 mg/kg caused no adverse weight change in C57
mice bearing B16F10 tumours [48]. When a PEGylated
melamine dendrimer [104] was injected into male C3H
mice in single doses up to 2.56 g/kg i.p. or 1.28 g/kg i.v.
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
no toxicity or mortality was seen although animals
were observed over a short period. Following i.v.
administration, this was 24 h and after i.p. injection it
was 48 h. No changes were observed in the blood
biochemistry (urea nitrogen levels or transaminases)
but a longer observation period is really necessary to
give greater insights. When preliminary experiments
were conducted to examine the in vivo toxicity of PEGpoly(ester) dendritic hybrids [30] compounds were
injected into mice as a single dose of 1.3 g/kg over 10 s
and animals were again observed over 24 h. Although
mortality resulted for one animal, other animals
survived and no changes in organ pathology were
observed after this short time.
4.3. Immunogenicity — a problem or an asset?
Due to their multivalency dendrimers are widely
viewed as a means to generate improved vaccines.
Peptide- (e.g., T cell epitope [79,129]), glyco- and
glycolipid-containing dendrimers have been described
[130]. However, unwanted immunogenicity (antigenicity) of dendrimers designed for other therapeutic
uses could prohibit their clinical development. Few
have systematically studied the cellular and humoural
effects of dendrimers. Further work is urgently needed
to ensure safe, chronic administration. In their studies
using cationic PAMAMs (generations 3, 5 and 7) in
rabbits Roberts et al. found no evidence of immunogenicity using two assays, an immunoprecipitation
assay and the Ouchterlony double diffusion assay [99].
5. Effect of dendrimers on cytokine and chemokine
release
Dendrimers can modulate cytokine and chemokine
release. This may prove a useful therapeutic tool, but,
as has been shown before for linear polymers, it can
produce catastrophic clinical toxicity. The synthetic
polyanion divinylether maleic anhydride (DIVEMA
or pyran copolymer) was known to induce interferon
release, activate macrophages and to promote tumour
cell killing, but it failed dramatically in early clinical
trials as an anticancer agent due to its severe toxicity
[131]. For many years the synthesis of glycodendrimers containing various surface moieties (including lactose, galactose, mannose, sialic acid and
2231
sulfated sugars (reviewed in Refs. [132,133]) have
been designed as multivalent anti-infective ligands
with potential anti-viral and antimicrobial properties.
A polysulfonate PAMAM (starting material PAMAM
generation 4) has also been shown able to block HIV1 and HIV-2 activity in vitro by inhibition of gp120
binding and inhibition of reverse transcriptase and
viral integrase activity [134]. Similarly, a polysulfated
galactose generation 5 DAB dendrimer (PS Gal
64mer), containing on average two sulfate groups
per galactose residue, has demonstrated ability to
inhibit HIV-1 infection of cultured U373-MAGICXCR4 and U373-MAGI-CCR5 [135]. Observation
that polysine dendrimers bearing naphthyl disodium
disulfonate terminal groups could reduce herpes
simplex virus (HSV-2) infection during vaginal
challenge in mice [136] provided the basis for clinical
testing of VivaGelk which is a generation 3 poly(llysine) dendrimer with a benhydrylamine core and 32
napthyl disodium disulfonate groups (SPL-2999) on
the surface [9].
It is however possible that dendrimer-related antiviral and antimicrobial activity seen in vivo may also
involve a component of immunomodulation. Vannucci
et al. [59] have shown that i.p. administration of Nacetyl-glucosamine-coated (GlcNAc8) PAMAM generation 1 glycodendrimers to mice bearing subcutaneous (s.c.) B16F10 melanoma model decreased tumour
growth and increased survival. Dendrimer administration increased CD69+ cells in spleen and tumour tissue.
Cytokines including interleukin (IL)-1h, interferon
(INF)-g, tumour necrosis factor (TNF)-a and IL-2
showed increased levels. Fluorescently labelled
GlcNAc8-PAMAM was found to localise in the liver,
kidney, spleen and lungs as well as tumour tissue.
We recently described PAMAM generation 3.5
dendrimers surface modified to contain either glucosamine or glucosamine 6-sulfate [78]. The PAMAM
generation 3.5-glucosamine dendrimers also showed
immunomodulatory properties inducing synthesis of
the pro-inflammatory chemokines MIP-1a, MIP-1h,
and IL-8, and the cytokines TNF-a, IL-1h, IL-6 from
human dendritic cells and macrophages. PAMAM
generation 3.5-glucosamine dendrimers was found to
be anti-angiogenic, thus by administering a combination of both dendrimers it was possible to reduce scar
tissue formation after glaucoma filtration surgery in an
animal model [78].
2232
R. Duncan, L. Izzo / Advanced Drug Delivery Reviews 57 (2005) 2215–2237
6. Conclusions
Interest in novel synthetic dendrimers and dendritic
polymers proposed for biomedical use continues to grow
exponentially (see reviews [19,37,136]). The architectures are ever more complex [72,137–140]. It is not
unusual to see sweeping statements in respect of
toxicological properties, e.g. bthese dendrimers are not
toxic nor immunogenicQ. This creates unhelpful dogma
and frequently the experimentation is not available to
back up claims in respect of the specific proposed use.
Less stringent toxicological tolerance limits are required
for in vitro dendrimer diagnostics such as the generation
2 PAMAM dendrimers functionalised with benzylpenicillinin used to detect IgE antibodies from sensitive
patients [141], and the luminescent [142] and fluorescent
dendrimers used as biochemical reagents or cellular
imaging tools [143]. For in vivo applications, there is a
need for carefully designed toxicology and toxicokinetic
studies for each dendrimer type, the protocols being
tailored to proposed clinical use. Lessons can be learned
from past clinical experience with other macromolecular
therapeutics (proteins, antibodies and polymer therapeutics) and particulate drug delivery systems including
liposomes, polymeric micelles and nanoparticles. The
several decades of clinical experience with these systems
is bringing an increased ability to predict potential
adverse reactions and an understanding of underlying
mechanisms. This knowledge can be particularly useful
in optimised design of multicomponent dendrimerbased systems that use a modular system to introduce
antibodies (e.g., Ref. [144]) or monomer units as
building blocks that already have a clinical history
(e.g., Ref. [145]). In conclusion, safe and efficacious
dendrimer-based delivery systems and therapeutics will
surely emerge, but only with and from rational chemical
design underpinned by a clear understanding of biological rationale.
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