Chapter 6
Chemical and EcotoxicologicalAssessment of Dendrimers inthe Aquatic Environment
Maria M. Ulaszewska*, M. Dolores Hernando{, Ana Ucles{, RobertoRosal}, Antonio Rodrıguez}, Eloy Garcia-Calvo* andAmadeo R. Fernandez-Alba*,{*Advanced Study Institute of Madrid, IMDEA-Agua, Parque Cientıfico Tecnologico, Madrid,
Spain{Department of Environment, Spanish National Institute for Agricultural and Food Research and
Technology (INIA), Madrid, Spain{Department of Analytical Chemistry, University of Almerıa, Almerıa, Spain}Department of Chemical Engineering, University of Alcala, Alcala de Henares, Spain
1. INTRODUCTION
Dendrimers are a relatively new class of polymeric materials which have gen-
erated much interest for innovative applications, mainly in biology, drug
delivery, catalytic processes or as imaging agents due to their layered struc-
tures and unique properties such as size, high degree of branching, internal
cavities, solubility controlled by the choice of surface groups, multivalency
and well-defined molecular weight.
The chemistry of dendrimers is very ‘elegant’ as their symmetric structures,
shapes and architecture resemble patterns found in nature such as snowflakes,
trees or neurons. Nowadays synthesis of dendrimers is a chemistry art in itself,
which allows the encapsulation inside complex organic structure of smaller
molecules such as drugs, pharmaceuticals or metals or the modification of the
macromolecule surface. Indeed, every year increases the amount of patents cov-
ering a wide range of applications, but at the same time, recent debate in scien-
tific circles is focussed on potential implications on health and environment.
Up to now, there is a serious lack of information about the levels of den-
drimers mostly used for pharmaceutical applications, such as PAMAM (poly-
amido amine) or PPI (polypropylene imine) in the environment; there is not
any improved analytical method which permits a sensitive detection and
Comprehensive Analytical Chemistry, Vol. 59. http://dx.doi.org/10.1016/B978-0-444-56328-6.00006-2# 2012 Elsevier B.V. All rights reserved. 197
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confirmation based on mass spectrometry. Since dendrimer and nanoscale
chemistry is one of the most rapidly expanding areas of modern chemistry,
we can expect further challenges due to these substances. This chapter
includes a section on analytical strategies in preparation, separation and charac-
terization of dendrimers. Other area of interest deals with recent advances in the
field of toxicity assessment, where despite the great interest in dendrimer chem-
istry, the few studies reported do not provide sufficient information for an effec-
tive risk evaluation. In the final section of this chapter, we present our approach
to analyze PAMAMdendrimer in natural waters by liquid chromatography–mass
spectrometry (LC–MS) using TOF and QTOF systems.
2. MOLECULAR STRUCTURE AND NOMENCLATUREOF DENDRIMERS
The term ‘dendrimer’ comes from the Greek word dendron, meaning ‘tree’, and
merosmeaning ‘part’; other synonymous terms are ‘cascademolecules’ or ‘arbor-
ols’ from the Latin word ‘arbor’ alsomeaning tree, obviously because the shape of
dendrimer resembles a tree, neuron or snow flake (Figure 1).
Dendrimers are large and highly branched polymers, the structure of
which is determined by three distinguishing architectural components: (1)
an initiator core, (2) an interior layer, composed of repeating units (genera-
tions), radially attached to the initiator core and (3) an exterior (terminal func-
tionality) attached to the outermost interior generation [1,2] (Figure 2).
A formal nomenclature was suggested for the DuPont SCION database
[3], but it is rarely used in the scientific literature. Due to the fact that
FIGURE 1 Different types of dendrimers and patterns in nature.
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dendrimers have very complex structure, the systematic name seldom is used
in common practice. Meijer’s naming system for PPI dendrimers follows this
scheme: Core-dendr-termini (i.e. DAB-dendr-(NH2)64) is a diaminobutane
core dendritic poly(propyleneimine) having 64 primary amine termini, also
named as DAB-dendr-(PA)64, where PA stands for polyamine [4]. Sigma-
Aldrich product names start with a symbol and added comments on core or
termini, for example, Family-(core)-Termini, followed by linear or ‘empirical’
formulas and formula weights of an ideal dendrimer (not the real average
molecular mass) [4].
Dendrimer diameters range from 1.5 nm for the first generation (G1) to
13.5 nm for G10, in the case of PAMAM dendrimer with ethylene diamine
(EDA) core (Table 1). Lower generation dendrimers usually have highly asym-
metric shapes with more open structures with respect to higher dendrimer gen-
erations. Themaximum size of a dendrimermolecule is limited to the generation
at which dendrimer becomes tightly packed, closed membrane-like structure.
Thus, when a critical branched state is reached, dendrimers cannot grow because
of a lack of space. This is called the ‘starburst effect’ [1,2]. Ideal dendrimer
structures are described with mathematical expressions [6]:
MW¼Mcþnc Mm NmG!1
� �
= Nm!1ð Þ� �
þMtnmG
!
; ð1Þ
where Mc, is the molar mass of the core; Mm, the molar mass of the branched
monomer; Mt, the molar mass of the terminal groups; nc, the core multiplicity;
nm, the branch-juncture multiplicity; G, the generation number. The increase
in the number of dendrimer terminal groups is consistent with the geometric
progression:
Z¼ nc∗nmG: ð2Þ
Branching units
Core moiety
Void spaces
Surface groups
G2
G1
G0
FIGURE 2 Structure of PAMAM dendrimer: core, branching units and surface groups.
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 199
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The molecular mass and the number of functional groups exponentially
increase with the generation number, but their diameter only increases linearly
by about 1 nm per generation. In the case of PAMAM dendrimers, half gen-
eration (G1.5, G2.5, G3.5, etc.) have carboxylic acid terminal groups and full
generations (G1, G2, G3, etc.) have amine or hydroxyl groups.
Dendrimers have some unique properties because of their globular shape
and the presence of internal cavities. Dendrimers can host a guest molecule
(i.e. hydrophobic or hydrophilic drugs) inside the interior cavities around
the core by host–guest interactions (see Figure 3A). The interactions between
host and guest molecules can take place not only in the interior of dendrimer
but also on the periphery. Binding groups responsible for encapsulation of
guest molecules in the interior and periphery are called endoreceptors and
exoreceptors, respectively.
The most investigated family of dendrimers is PAMAM, built up by polia-
mide branches with tertiary amines as focal points. For the first time PAMAM
dendrimers were synthesized by Tomalia and co-workers in the mid-1980s
[1,2], and up to day their structures have achieved many modifications of ter-
minal groups and have found a wide range of uses.
TABLE 1 Molecular Mass and Number of Terminal Groups for Different
Generations of PAMAM Dendrimers (According to Equations 1 and 2)
Generation
Ammonia Coreb EDA Corea
MolecularMass
Number of
TerminalGroups
Diameter(nm)
MolecularMass
Number of
TerminalGroups
Diameter(nm)
0 359 3 1.08 516 4 1.5
1 1043 6 1.58 1428 8 2.2
2 2411 12 2.20 3252 16 2.9
3 5147 24 3.10 6900 32 3.6
4 10,619 48 4.0 14,196 64 4.5
5 21,563 96 5.30 28,788 128 5.4
6 43,451 192 6.70 57,972 256 6.7
7 87,227 384 8.0 116,340 512 8.1
8 174,779 768 9.20 233,076 1024 9.7
9 349,883 1536 10.5 466,548 2048 11.4
10 700,091 3072 12.4 933,492 4096 13.5
aFrom Ref. [11].bFrom Ref. [5].
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A second group of well-investigated dendrimers with widespread applica-
tion in materials science and biology is PPI, created by Vogtle and co-workers
and produced at large scale by DSM (Dutch State Mines) [7]. PPI are polyalk-
ylamines having primary amines as end groups, the interior part of the mole-
cule consisting of tertiary tris-propylene amines. PPI dendrimers are
sometimes called POPAM, which stands for polypropylene imine or DAB
dendrimers, where DAB refers to the core structure diaminobutane.
In 1990, Frechet synthesized aromatic dendrimers using 1,1,1-tris(4-hydroxy-
phenyl) ethane as core material and benzylic bromide and 3,5-dihydroxybenzyl
alcohol as branching materials [8]. In the early 1990s, phenylacetylene dendri-
mers were created by Jeffrey and Moore. These types of dendrimers are called
Frechet type, and can be symmetric or asymmetric consisting of two or three parts
of segmental dendrons (see Figure 3B). Their surface usually contains carboxylic
acid groups serving as anchoring point for further functionalization. In recent
years, dendrimers with different designed functionalities have become objects
of particular academic and practical interest because of their unique superb-
ranched architectures, high densities of peripheral functionalities, symmetrical
shapes and monodispersity.
3. PHYSICAL AND CHEMICAL PROPERTIES OF DENDRIMERSIN SOLUTION
Physico-chemical properties of dendrimers depend on the generation number,
surface functionalities and core structure. In solution, dendrimer can exist as a
tightly packed ball, and this can have a great impact on their rheological proper-
ties. Dendrimer solutions exhibit significantly lower viscosity than linear poly-
mers [9] which does not increase linearly with molecular weight but shows a
A B
Small
guest
Small
guestSmall
guest
Small
guest
Small
guest
Small
guest
Small
guest
Largeguest
Largeguest
Largeguest
Largeguest
FIGURE 3 (A) Scheme of a host–guest system for PAMAM dendrimers. Internal cavities can
host small and large molecules; (B) Frechet-type of aromatic dendrimers with zinc porphyrin
(ZP) core.
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 201
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maximum at the fourth generation to decline thereafter [10]. Such behaviour is
caused by the way in which dendrimer shape changes with generation num-
ber—lower generation dendrimers are large enough to be spherical and have
enormous surface areas in relation to volume; high-generation dendrimer have
more compact spherical shape and are tightly packed. Dendrimers’ solubility is
strongly influenced by the nature of surface groups. Dendrimers with a hydro-
phobic interior and hydrophilic surface are called unimolecular micelles because
they are able to solubilize hydrophobic molecules in aqueous solutions, while
dendrimers having hydrophobic end groups are soluble in non-polar solvents.
Figure 4 shows correlation between dendrimer generations and several properties
such as density, surface area viscosity and refractive index.
A fundamental property for understanding environmental fate and beha-
viour of contaminants in aquatic systems is n-octanol/ water partition coeffi-
cient. In case of PAMAM molecule with surface amine groups and EDA
core (i.e. PAMAM G4) at acidic pH (<5), all the primary and tertiary amine
groups of the dendrimer are protonated. At neutral pH 7.0, only the primary
amine groups are protonated. Conversely, all the amine groups become unpro-
tonated at basic pH (>10.0). PAMAM dendrimers have higher log Kow value
(!2.15) at acidic pH 4 than at neutral or basic pH, and there is no significant
change in the log Kow value when pH changes from neutral to basic [12].
For dendrimer with the same structural core (EDA core and spherical groups),
log Kow gradually decreases with generation from G1 to G4 (from !1.53
to !2.36) followed by slight increase at G5 (log Kow of !2.16) at room tem-
perature and pH of 7.4. PAMAM dendrimers with terminal NH2 groups have
LGD MGD
Generation
Intrinsic viscosity (h)
Surface area/Head group (Z)
Refractive index
Density (d)
0 1 2 3 4 5 6 7 8 9
HGD
FIGURE 4 Calculated and measured properties of PAMAM dendrimer in solution (LGD, MGD,
and HGD—Low- Medium- and High-Generation Dendrimer) [11].
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negative log Kow values thereby suggesting their hydrophilicity [12]. For
PAMAM G6 and G8, the formation of stable emulsions suggests they prefer
to partition at the O/W interface.
Giri and co-workers studied the influence of different terminal groups for
PAMAM dendrimer G4 with EDA core on Kow: cationic (amine, amidoethy-
lethanolamine), anionic (carboxylate, succinamic acid and pyrrolidinone) and
neutral (amidoethanol and tris(hydroxymethyl) amidomethane (tris)). The
PAMAM G4 with tris terminal groups has the highest (less negative) log
Kow (!1.39) [12]. In the same study, the authors tested the influence of differ-
ent cores on the log Kow values (see Table 2).
3.1 Effect of Various Factors on Dendrimer Chemistry:Conformational Changes
3.1.1 Effect of pH
The structure of dendrimers having basic surface groups (i.e. amino-terminated
PAMAM or PPI) at low pH (<4) is highly ordered and shows an extended
TABLE 2 Influence of Different Cores and Terminal Groups on Log Kow of
PAMAM Dendrimers at pH 7.4 and at Room Temperature [12]
Generation Core Terminal Group log Kow
Influence of different terminal groups on log Kow
4 EDA Amine !2.38%0.02
4 EDA Amidoethanol !2.47
4 EDA Succinamic acid !2.53%0.06
3.5 EDA Sodium carboxylate !2.33
4 EDA tris(hydroxymethyl) aminomethane !1.39
4 EDA Pyrrolidinone !2.45%0.08
4 EDA Amidoethylethanolamine !2.54
Influence of different cores on log Kow
4 EDA Amine !2.38%0.02
4 DAB Amine !2.35
4 DAH Amine !2.25
4 DAD Amine !2.33
4 Cyst Amine !2.62
EDA, ethylene diamine; DAB, diaminobutane; DAH, diaminohexane; DAD, diaminododecane;Cyst, cystamine.
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 203
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conformation due to the electrostatic repulsion between positively charged
ammonium groups [13]. At neutral pH, back-folding occurs which may be
caused by hydrogen bonding between the unchanged tertiary amines in the inte-
rior and the positively charged surface amines. At higher pH (pH>10),
PAMAM dendrimers adopt a globular shape as the charge of the molecule
becomes neutral. At this pH, the conformation has a higher degree of back-
folding as a consequence of the weak ‘inter-dendron’ repulsive forces
[14,15]. Dendrimers with acidic end groups (i.e. PPI with carboxylic acid
spherical groups) behave a bit different from the previously described dendri-
mers. SANS (small angle neutron scattering) and NMR (nuclear magnetic res-
onance) measurements showed that at pH 2, an electrostatic repulsion takes
place between the positively charged protonated tertiary amines, leading to a
larger radius of the core. By approaching more neutral levels, the amount of
positively charged amines equals the amount of negatively charged carboxylic
groups resulting in a ‘dense core’ conformation more prone to back-folding.
Conformation in back-folding occurs as a consequence of electrostatic interac-
tions between the negatively charged surface carboxy-groups and the positively
charged tertiary amines in the inner shells of the dendrimer [14,16]. At basic
pH (close to 11), the electrostatic repulsion between the negative charged
forces of the surface groups again results in more extended conformation with
a highly expanded surface area [14] (see Figure 5).
3.1.2 Effect of Solvent
Solvent polarity has a great influence on the structure of dendrimers and is a
very important parameter when investigating their conformational state.
‘Dense shell packing’ favoured by e.gattractive forces between surfacegroups—highly ordered state.
Back-folded conformation. Favoured bye.g weak forces between surfacegroups—less ordered state.
FIGURE 5 Schematic representation of the consequence of back-folding resulting in an
increased molecular density in the interior of a dendrimer.
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In general, dendrimers show a larger extent of back-folding with decreasing
solvent properties, that is decreasing solvation. In comparison to higher gen-
eration dendrimers, the lower generations show a higher tendency towards
back-folding as a result of poorer solvation [14,17].
Experiments carried out with amino-terminated PPI dendrimer and two
kind of solvents, benzene and chloroform, showed that non-polar solvent
(benzene) poorly solvates the dendrons causing interactions between segments
and back-folding. The use of a weakly acidic solvent such as chloroform,
which acts as hydrogen donor for the inner amines, resulted in an extended
conformation for PPI [18]. For amino-terminated PAMAM, non-polar aprotic
solvents also induce higher molecular density in the core region as a conse-
quence of back-folding effect, while polar solvents induce a higher molecular
density on dendrimer surface. The response is rather different for Frechet-type
dendrimers. In an experiment with toluene, the hydrodynamical volume of a
PAMAM dendrimer increased with stronger effect observed for the lower
generations. This may be a consequence of the more open structure of low-
generation dendrimers which allow solvent molecules to penetrate inside the
dendrimer [14,19].
3.1.3 Effect of Salts
At high concentration of salts, the tendency towards back-folding also
increases and a dense core dendritic structure is formed. At low concentration,
dendrimers adopt an extended conformation to minimize charge repulsion
within the structure [20]. The effect is somewhat similar to that observed at
different pH conditions.
3.1.4 Effect of Concentration
The effect of the concentration has been described by experiments performed
with PPI G4 and G5 dendrimers [21]. In a polar solvent, methanol at increas-
ing concentrations, the structure of the two generations of PPI becomes
increasingly contracted. The molecular contraction might contribute to mini-
mize the repulsive forces among the functional groups and thus, to exhibit a
more tight intramolecular packing.
4. SYNTHESIS AND APPLICATIONS OF DENDRIMERS
Since the early work of Tomalia et al. [1,2], two main strategies have been
developed for the synthesis of dendrimers, namely divergent and convergent
approaches (Figure 6).
In the divergent approach, the branching units are successively attached to
the core molecule so that the number of peripheral groups depends on branch-
ing multiplicity (Figure 10). This divergent approach is currently the preferred
commercial strategy used by worldwide producers [5].
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 205
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In convergent synthesis, reactions proceed inwards from the surface of the
dendrimer eventually attaching a core to give a complete dendrimer [5]
(Figures 6 and 7). The convergent strategy is preferred for the synthesis of
asymmetric dendrimers in which different segments are coupled to create het-
erogeneous morphologies [14,23]. Other approaches are based on dendrimer
self-assembly properties. Divergent synthesis by self-assembly was proposed
by Nilsen et al. [24] who used oligonucleotides as building blocks followed
by cross-binding to stabilize the self-assembled dendrimer. This approach
has been proposed for the synthesis of scaffolds for biomolecules to be used
in diagnostics [14].
Recently, two newbreakthrough approaches in dendrimer synthesis have been
reported. The first one, called ‘lego’ chemistry, uses highly functionalized cores
and branched monomers to create phosphorus-based dendrimers (Figure 8A)
[25]. Several variations have been developed thereafter, allowing multiplication
of the number of terminal surface groups from 48 to 250 in one single step. These
dendrimers require just one step per generation, used a minimum volume of sol-
vent and allow facile purification [26,28]. The second approach is based on ‘click’
chemistry bywhich several authors reported the use ofCu(I)-catalyzed reaction of
1,2,3-triazoles from azides and alkynes to produce dendrimers with various sur-
face groups in high purity and yield (Figure 8B) [27,29,30].
The well-defined particle size and shape of dendrimers combined with
their ‘guest–host’ properties give reason for their broad interest in pharmaco-
logical and imaging applications, many of them due to their ability to cross
cell membranes, so reducing the risk of premature elimination from the body.
Dendrimers are also in use in classic technologies such as coatings and
inks due to the physical properties associated with their spherical molecular
shape. Either in nanotechnology or traditional technology, the versatility of
ConvergentDivergent
FIGURE 6 Divergent versus convergent strategies. The blue dots mark the ‘functionalizing
sites’.
Analysis and Risk of Nanomaterials206
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dendrimers is behind the high number of patents, which are currently issued
for the most diverse applications [31].
Dendrimer use in drug delivery is based on their ability to host–guest
molecules either in the interior of dendrimers or on the periphery
(Figure 3A) [5]. Delivery approaches include: (i) drug covalently attached
to the periphery (prodrugs), (ii) drug coordinated to the outer functional
groups via ionic interactions and (iii) drug encapsulated through the formation
of a dendrimer–drug supramolecular assembly. Most bioactive drugs have
hydrophobic moieties, which result in low water solubility and hinder effi-
cient delivery. Dendrimers designed to be water soluble and biocompatible
have been used for the delivery of non-steroidal anti-inflammatory drugs as
well as other compounds such as antiviral, antimicrobial, anticancer or antihy-
pertensive drugs [32–35]. It has also been shown that the surface-modified
forms of dendrimers may exhibit a greater potential as solubility enhancers
for insoluble drugs [36]. Dendrimers can serve as effective pro-drug scaffolds
with the drug molecules being attached through a linkage, which activates
in vivo once reaching the delivery site [37].
(a) H2C=CHCO2Me (a)
(b) Raney cobalt
(b) Raney cobalt
(a)
(b) H2NCH2CH2NH2
Repeat steps (a) & (b)
FIGURE 7 Synthetic schemes of poly(amidoamine) and poly(propyleneimine) dendrimers [22].
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 207
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Molecular imaging is possible through the attachment of fluorophores
(for optical), metal chelates (for fluorescence, magnetic resonance imaging,
positron emission tomography and single photon emission computed tomog-
raphy) or with a switch activated by a physiological process [37–39]. See
lanthanide ions with poly(aryl ether) dendritic ligands in Figure 5B. Recent
innovations in dendrimer research have increased agent directibility and
new synthetic chemistry approaches have increased efficiency of production
RN
N+
N
ClCl
A
B
X
Cl
X
Cl
Cl
X
X
coreTriazole
dendrimer G3
Triazole
dendrimer G4core
N3
N3
R-X-(G1)-Cl
R-X-(G2)-Cl
R-X-(G1)-N3
R-X-(G2)-N3
R-X-(G1)-N3
R-X-(G2)-N3 R-X-(G3)-N3
R-X-(G4)-N3
R-X-(G3)-N3
R-X-(G4)-N3R-X-(G3)-N3
3 6 12
1 step
2 equiv.
2 equiv.
2 equiv.
2 equiv.
+
+
+
+
a
a
a b
b
a
a
+
+ba
b
1 step 1 step
−
N
N
X
N
R
N
N
N
R
N
N
X
N
R
N
N
N
R
N
N
X
N
R
N
N
N
R
N
N
X
NN
N
N
N
N
X
N
R
N
N
N
R
N
N
X
N
R
N
N
N
R
N
N
X
NN
N
N
N
N
X
N
R
N
N
N
R
Cl
FIGURE 8 (A) Divergent approach using ‘lego’ chemistry towards highly functionalized phos-
phorous dendrimers. (B) Convergent approach towards triazole dendrimers using ‘click’ chemis-
try: (a) CuSO4 (5 mol%), sodium ascorbate (10 mol%), H2O/tBuOH (1:1); (b) 1.5 equiv. NaN3,
CH3COCH3/H2O (4:1), 60 &C, 1–3 h [25–27].
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as well as led to the creation of novel dendrimer-based contrast agents
[40,41].
Concerning catalytic applications, dendrimers can behave both, as catalyt-
ically active species and soluble supports [42–47]. One advantage of dendritic
supports is the easy separation from the reaction mixture using ultrafiltration,
dialysis or ultracentrifugation [48]. Additionally, the high density of catalyti-
cally active species at the periphery of the dendritic support is expected to
lead to synergistic effects and stereo selectivity [49,50].
The use of PAMAM dendrimers in personal care products has been
recently reported as active component of deodorants and self-tanning cos-
metics [51].
5. TOXICITY AND ECOTOXICITY
The possible therapeutic use of dendrimers has recently generated a consider-
able interest among scientists. Their bioactivity suggests their use in a number
of cases, which cover from bone mineralization to the formulation of antican-
cer drugs [52]. One of the most explored applications is the use of cationic
dendrimers such as antimicrobial agents. Glycodendrimers with surface car-
bohydrates can be linked to carbohydrate targets on the cell surface, which
act as receptor for viruses and, therefore, can prevent infections [53]. Chen
and Cooper [54] suggested the use of cationic dendrimers as antimicrobial
agents based on their electrostatic interaction with negatively charged bacte-
ria. Dendrimers are also able to prevent replication of the viral genome by
inhibiting several viral enzymes [55]. In fact, the only commercial application
of dendrimers is by the moment VivaGel, an antiviral agent for the treatment
of sexually transmitted diseases launched by Starpharma. The company
recently announced successful results of phase II clinical trials performed in
collaboration with the U.S. National Institute for Allergy & Infectious Dis-
eases. The active component of VivaGel is the proprietary polylysine dendri-
mer SPL-7013, which is aimed to bind to surface proteins on HIV, to prevent
the virus from infecting human T cells. The product is also expected to pre-
vent other sexually transmitted diseases. Dendrimers have also shown poten-
tial application as therapeutics against cancer; glycodendrimers have emerged
as convincing targets for carbohydrate-binding proteins on tumour cells
increasing survival in mice induced with a model melanoma [56]. Starpharma
recently launched a commercial research reagent kit called NanoJuice Trans-
fection Kit for transporting DNA into cells using proprietary dendrimer tech-
nology from Starpharma’s subsidiary Dendritic Nanotechnologies. Stratus CS
Acute Care DDMR Assay from Siemens Healthcare Diagnostics is a labora-
tory immunoassay for cardiac diagnosis, which uses a dendrimer-linked
monoclonal antibody [57].
There are many research articles on dendrimers covering the drug delivery
potential of these compounds when acting as nanocarriers. From this point of
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 209
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view, dendrimers belong to a class of macromolecular therapeutics that
includes other particulate drug delivery systems such as polymeric micelles
and liposomes. A key point is that PAMAM dendrimers as oral drug carriers
is the ability of certain PAMAM-drug systems to permeate the epithelial
barrier of the gut [58] also demonstrated that cationic and anionic PAMAM
dendrimers enter cells through clathrin-dependent endocytosis mechanism
to be rapidly trafficked to endosomal and lysosomal compartments. The deliv-
ery of oligonucleotides using synthetic nanomaterials as an alternative to
viral carriers has been a topic of study during recent years [59]. Baigude
et al. [60] used a positively charged polylysine-based dendrimer with unsatu-
rated hydrocarbon side chains for RNA interference with results comparable
to a commercial transfection reagent. Nanoscale drug delivery materials
are expected to not only allow targeted intracellular delivery but also
provide imaging capability for following the uptake of the material [61].
When acting as a drug carrier, the reduction of drug toxicity is an important
issue, particularly in anticancer drugs, many of which are known to be toxic.
For example, Neerman et al. [62] showed that melamine dendrimers can
reduce the organ toxicity of solubilized cancer drugs administered by intraper-
itoneal injection.
Even if dendrimers can reduce the toxicity of carried drugs, the unwanted
toxic response to dendrimers themselves is a cause for concern and could limit
their clinical development. In spite of the large number of biocompatible
formulations under study, the safety of long-term use is still to be assessed
[52]. It has been suggested that the clinical experience with more classical
macromolecular or polymer-derived therapeutics can be used as a guide for
dendritic nanomedicine [63]. For example, Naha et al. [64] observed that poly
N-isopropylacrylamide (PNIPAM) nanoparticles were internalized and loca-
lized in lysosomes of two different mammalian cell lines but no toxic effects
were observed at any concentration, proving the excellent biocompatibility of
this material. Clinical testing, however, would require careful toxicokinetic
and toxicodynamic studies, but the studies evaluating the possible adverse
effects of dendrimers on humans offer results that are fragmentary and often
contradictory [65].
An important work has been already performed concerning the toxicity of
dendrimers to cell lines from higher organisms. Mukherjee et al. [66] sug-
gested a connection between surface charge of G4–G6 PAMAM dendrimers
and their toxicity to two mammalian cell lines. They showed that the adsorp-
tion of proteins on nanoparticle surface resulted in a dramatic change in
z-potential. This kind of activity–structure relationship is still poorly
understood and requires further work to be enlightened. Lee et al. [67]
observed down-regulation of mitochondrial DNA-encoded genes involved
in the maintenance of mitochondrial membrane potential for human lung
cells-exposed PAMAM dendrimers (1,4-diaminobutane core, G4-NH2). The
Analysis and Risk of Nanomaterials210
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authors observed damage in mitochondria and a decrease of cell viability with
activation of caspases 3 and 9 resulting in apoptosis. Nomami et al. [68]
showed that the toxicity of self-assembled PAMAM/antisense nanoparticles
to human breast cancer depends on both the PAMAM concentration and
generation. To minimize the toxicity of dendrimers, Jain et al. [69] suggested
masking of peripheral charge by surface modification. Biocompatible dendri-
mers can be produced by employing a biodegradable core and by neutralization
of the external charge, for example, PEGylation or peptide conjugation.
Ortega et al. [70] prepared biocompatible carbosilane dendrimers with mannose
peripheral groups by deacetilation of a precursor and proved that they are
non-toxic for dendritic cells.
Concerning animal models, Li et al. [71] studied the molecular link
between exposure and lung damage in mice using a cationic PAMAM from
Starburst. They observed toxicity due to acute lung injury in vivo because
PAMAM triggers autophagic cell death by deregulating a signalling pathway.
The damage would be reversed by using an autophagy inhibitor. Chauhan
et al. [72] studied the intraperitoneal toxicity of G4-NH2 and !!OH PAMAM
dendrimers in Swiss albino mice. They found certain reduction in glucose
levels in the high-NH2 dose group probably indicating the interference with
glucose metabolism as well as toxic effects on kidney and liver which recov-
ered normal values after ceasing exposure. On the other hand, Chen et al. [73]
found no toxicity for pegylated dendrimers at doses up to 2.56 g/kg i.p. injec-
tion in male CH3 mice whereas100% mortality was observed for cationic den-
drimers at 160 mg/kg [74].
The available information concerning toxicity of dendrimers on non-target
organisms is still very limited. Up to date, only a few studies have been per-
formed to assess the ecotoxicity of dendrimers, the most complete being per-
formed by Naha et al. [75] using PAMAM dendrimers of generations G4, G5
and G6. They demonstrated a significant toxicity, which increased with
increasing dendrimer generation with median effect values ranging from
0.129 (7.4 mg/l, Daphnia magna, 48 h, G6) to 16.30 mM (231.5 mg/l, Vibrio
fischeri, 5 min, G4). They also suggested a positive correlation between
EC50 and the change in z-potential, which takes place when dendrimers are
put in the corresponding assay media rather than to their actual z-potential.
This kind of quantitative structure–activity relationship is still to be proved
in larger assays, but may suggest an interaction with the medium, which could
determine the toxic response.
Successive generations present a larger surface area for interaction and
have been associated with higher toxicity [75]. Petit et al. [76] investigated
the toxicity of G2, G4 and G5 PAMAM dendrimers to the green alga
Chlamydomonas reinhardtii. Using cell viability measured from esterase
activity as endpoint, they found that toxicity decreased with dendrimer gen-
eration number (2 mg/l for G2, 3 mg/l for G4 and 5 mg/l for G5) when
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 211
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expressed in mass concentration units. The results in molar concentration
units indicated, however, a toxicity increase with generation number
(Table 3). Petit et al. [76] observed the stimulation of the photosynthetic
activity and the increase of total chlorophyll content of algae exposed to
G2 and G4 PAMAM dendrimers. The reason is difficult to assess, but it is
probably due to the activation of the photosynthetic electron transport of
Photosystem II.
The toxicity of PAMAM dendrimers to bacteria has been also studied by
Mortimer et al. [78] who obtained 30-min EC50 values of 631 mg/l
(194 mM, G2) and 775 mg/l (27 mM, G5). The toxicity increased for higher
generations in agreement with Naha et al. [75], but the EC50 values obtained
were considerably higher. V. fischeri was quite sensitive to other carbon-
based nanoparticles. Naha et al. [79] reported 5-min EC50 of 40.5 and
25.7 mg/l for N-isopropylacrylamide-co-N-tert-butylacrylamide in 65:35
and 50:50 copolymers (molecular weights not given), respectively. Heiden
et al. [80] using zebrafish embryos have studied the toxicity towards a
higher ecotoxicologically relevant organism. They determined the toxicity
of G3.5 and G4 PAMAM dendrimers, including some dendrimers bioconju-
gated with the Arg-Gly-Asp (RGD) sequence, which is known to play a role
in cell recognition system. They found that G4 dendrimers were toxic
towards growth and development of zebrafish embryos, but G3.5 dendri-
mers, with carboxylic acid terminal functional groups, did not exhibit toxic-
ity. PAMAM dendrimers with RGD reduced its cytotoxicity with respect to
unmodified G4.
Suarez et al. [77] recently published the toxicity of PAMAM G1 and G4
dendrimers with surface amino groups as well as G4-OH with terminal
hydroxyl groups. The toxicity to Pseudokirchneriella subcapitata, with 72-
h growth inhibition determined as indicated in OECD TG 201 (open system)
was followed by measuring chlorophyll fluorescence. The toxicity was partic-
ularly high for G4-NH2 with EC50 1.41 mg/l (99 nM) whereas the median
effect value for the G1-NH2 was 5.86 mg/l (4.1 mM). This result supports
others’ findings indicating increased toxicity for higher dendrimer generation,
probably as a consequence of a larger surface area for interaction with living
organisms [75]. Suarez et al. measured (as z-potential) relatively high positive
charges for G4-NH2 dendrimers (þ26.1%1.6 mV). This positive charge is
relevant for toxicity because algal cell membranes possess large negatively
charged domains, which should favour particle–cell interaction. The toxicity
of the negatively charged, hydroxyl-terminated G4-OH dendrimer was
much lower (EC50>40 mg/l or 2.8 mM). Suarez et al. also showed that no
observed effect concentration determined as EC10, was considerably low
(0.21 mg/l or 15 nM for G4-NH2 and 0.43 mg/l or 30 nM for G4-OH) even
for hydroxyl-terminated dendrimers.
The effect of dendrimers must take into account the mechanisms of inter-
nalization, which result in a direct toxic response and which should be
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TABLE 3 Literature Data Reported for the Ecotoxicity of PAMAM
Dendrimers
Dendrimer Type and Test Species EC50 in mM References
G1 ethylenediamine core (8 NH2 surface groups)
Pseudokirchneriella subcapitata (72 h) 4.1 (3.8–4.4) [77]
G2 1,4-diaminobutane core (16 NH2 surface groups)
Chlamydomonas reinhardtii (72 h) 0.590 (0.548–0.630) [76]
G2 ethylenediamine core (16 NH2 surface groups)
Vibrio fischeri (30 min) 194 (66–321) [78]
G4 1,4-diaminobutane core (64 NH2 surface groups)
Chlamydomonas reinhardtii (72 h) 0.208 (0.180–0.237) [76]
G4 ethylenediamine core (64 NH2 surface groups)
Vibrio fischeri (5 min)Vibrio fischeri (15 min)Vibrio fischeri (30 min)Daphnia magna (24 h)Daphnia magna (48 h)Thamnocephalus platyurus (24 h)
16.306.173.111.130.682.90
[79]
Danio rerio embryios (24 h post-fertilization)Danio rerio embryios (72 h post-fertilization)Danio rerio embryios (120 h post-fertilization)
1.0 (0.9–1.2)0.6 (0.5–0.6)0.4 (0.3–0.4)
[80]
Pseudokirchneriella subcapitata (72 h) 0.099 (0.096–0.106) [77]
G4-RGD ethylenediamine core (64 NH2 surface groups)
Danio rerio embryios (120 h post fertilization) 4.1 [80]
G4 ethylenediamine core (64 OH surface groups)
Pseudokirchneriella subcapitata (72 h) >2.8 [77]
G5 1,4-diaminobutane core (128 NH2 surface groups)
Chlamydomonas reinhardtii (72 h) 0.167 (0.158–0.171) [76]
G5 ethylenediamine core (128 NH2 surface groups)
Vibrio fischeri (5 min)Vibrio fischeri (15 min)Vibrio fischeri (30 min)Daphnia magna (24 h)Daphnia magna (48 h)Thamnocephalus platyurus (24 h)
15.185.081.640.720.271.81
[79]
Vibrio fischeri (30 min) 27 (14–40) [78]
continued
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 213
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strongly generation-dependent. It has been recognized that the interaction of
dendrimers with lipid bilayers is on the basis of their toxicity [81]. Mecke
et al. [81] also found that polycationic nanoparticles might induce the forma-
tion of holes at least in the crystalline phase of bilayers. The resulting increase
in membrane permeability is a possible mechanism for nanoparticle internali-
zation [19]. Much research is still to be conducted to elucidate the underlying
mechanism of toxic response including nanogenotoxicity, the relationship of
toxicity with molecular structure. Currently, the environmental risk assess-
ment of nanomaterials is seriously hampered by the lack of information. It
is crucial to assess not only the toxicity of nanomaterials but also their envi-
ronmental fate considering degradation processes and the generation of trans-
formation products. New developments in analytical methods are required to
detect environmental concentrations the estimation of which is otherwise hin-
dered by the lack of knowledge about the rates of release of nanomaterials to
the environment and the theoretical background necessary to predict actual
concentrations from release rates.
6. ANALYTICAL CHARACTERIZATION
Analytical characterization of dendrimers presents important difficulties,
because they are just between molecular chemistry and polymer chemistry.
They belong to the molecular chemistry world due to their ‘step-by-step’ syn-
thesis and the area of polymer chemistry because of the repetitive structure
made of highly branched monomers. Till now, the methods involved in prep-
aration, separation and characterization of dendrimer structures reported in the
literature are mainly based on microscopy, scattering, electrophoresis, chro-
matographic separation, spectroscopy and spectrometry. Table 4 includes per-
formance characteristics of techniques applied in the elucidation of dendrimer
structures.
TABLE 3 Literature Data Reported for the Ecotoxicity of PAMAM
Dendrimers—Cont’d
Dendrimer Type and Test Species EC50 in mM References
G6 ethylenediamine core (256 NH2 surface groups)
Vibrio fischeri (5 min)Vibrio fischeri (15 min)Vibrio fischeri (30 min)Daphnia magna (24 h)Daphnia magna (48 h)Thamnocephalus platyurus (24 h)
4.801.640.830.320.131.11
[79]
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TABLE4SummaryofIm
aging,Separationan
dSpectrometryTech
niquesAppliedto
Characterize
DendrimerStructures
Imag
ing
Tech
nique
Size
Ran
ge
(nm)
Param
eter
Separation
Tech
nique
Size
Ran
ge
(nm)
Param
eter
Spectrometry
Tech
nique
SizeRan
ge
(nm
orDa)
Param
eter
TEM
1–1
000
Size,shap
eHPLC
<10
Size
,physic-
chem
ical
properties
MALD
I-MS
1–1
300kD
aMass,co
mp.
SEM
5–1
000
Size,
shap
e,structure
SEC
0.5–1
0Size
ESI-MS
HPLC
–MS
0.02–2
000Da
Mass,co
mp.
ESEM
40–1
000
Size,
shap
e,structure
CE
1–1
000
Size
,ch
arge
AFM
0.5–1
000
Size,
shap
e,structure
GEM
MA
2.5–1
000
Elec
trophoresis
mobility
HPLC
–QTOF
0.02–4
0,000Da
Mass,co
mp.
Auth
or's p
ers
onal copy
6.1 Spectroscopy and Spectrometry: ESI-MS, MALDI-MS,QIT, NMR, UV–Vis
MS plays an important role in dendrimer chemistry. It allows to study the
fragmentation mechanisms of dendrimers and to distinguish defects formed
during the synthesis [82,83]. It serves also to study the properties of the den-
drimer molecules or their host–guest complexes in the gas phase, providing
valuable new insight under environment-free conditions, which cannot easily
be studied in solution. MS is much more than merely a tool to evaluate the
effectiveness of a synthesis strategy to generate defect-free dendrimers and
its potential for dendrimer chemistry has not yet fully been appreciated [83].
Electro spray ionization (ESI) and matrix-assisted laser desorption ionization
(MALDI) have been successfully applied thanks to their ability to form stable
multicharged dendrimer species [84,85]. By ESI-MS, Weener and co-workers
studied the fragmentation mechanisms of PAMAM dendrimer from genera-
tion 1 to 5 which were explained by nucleophilic displacement reaction, pro-
ton shifts and rearrangements. They demonstrated that fragmentation was
generated by nucleophilic attack of a nitrogen on an alpha carbon-protonated
nitrogen and depending on the site of protonation as well as on the basicity of
the nitrogen atoms [86,87].
The MALDI technique was first presented in 1988 and it has been a major
breakthrough for the mass spectrometric analysis of large molecules
(Mw>2 kDa) [88,89]. It has been used for characterizing the purity of aro-
matic polyesters [90], polybenzylacetylenes [91], PAMAM [92], PBzE [93],
silicon dendrimers [94,95] or phosphorus dendrimers [26]. Mass spectral data
on dendrimers by MALDI needs to be interpreted with care, since it may be
misleading in the sense that falsely negative results might be obtained.
Schalley and Baytekin examined MALDI and ESI-MS to investigate their
possibilities and limitation in dendrimer characterization [82,96]. Indeed, both
techniques have some limitations, and may yield a picture which does not pre-
cisely reflect the real sample composition. During analysis of persulphony-
lated POMAM dendrimers, MALDI turns out to induce reactions within the
matrix during the ionization processes and lead to the same products which
would be formed in an incomplete synthesis. For better understanding of this
issue, Baytekin and co-workers performed an experiment with different types
of POMAM dendrimers in order to investigate the presence of defects on the
dendrimer surface [82]. In the case of dendrimers bearing sulphonamide
groups in their periphery, the ESI mass spectra provided evidence of sample
purity, while MALDI gave signals associated with structural defects. The
cause was associated to the thermal reactions occurring during the ionization
within the matrix, and not to the synthesis which proceeds cleanly to the
desired products. In another example, ESI mass spectra of POMAM dendri-
mer with amine surface were also related to the presence of a high abundance
of a new type of defects which, by contrast, were detected in neither the
Analysis and Risk of Nanomaterials216
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MALDI mass spectra nor the 1H or 13C NMR spectra. Subbi and co-workers
examined different matrices for the analysis of PAMAM dendrimer by
MALDI: 2,5-dihydroxybenzoic acid (DHB), 4-hydroxy-3-methoxycinnamic
acid (FER), a-cyano-4-hydroxycinnamic acid (ACH), 2,4,6-trihydroxyaceto-
phenone (THAP) and 3-hydroxypicolinic acid (HPA). Of these, DHB
was the softest matrix and ACH produced significant fragment intensity
already at MALDI threshold, FER and THAP being in between. HPA was
not a convenient matrix for dendrimers and produced a specific fragmentation
pattern [97].
MALDI coupled with a time-of-flight mass-spectrometer (TOF-MS) is
nowadays one of the most frequently used techniques for dendrimer exact
mass determination; mass accuracy, sensitivity and high molecular weight
range are the most important features [98]. On the other hand, the disadvan-
tages of this technique are rather high instrumentation costs, the search of
optimal matrix and the sample preparation method. ESI-MS has the advantage
of lower instrumentation costs and the generation of highly charged species.
For the determination of the multiply charged ions, however, the exact charge
state could be necessary. For that, the peaks that represent adjacent charge
states would have to be resolved, being especially difficult for many large
molecules (i.e. PAMAM G6) [83,99].
Other spectrometry techniques such as chemical ionization or fast atom
bombardment might be also used, but only for small dendrimers <3000 Da
[8]. Coupling of ESI or chemical ionization with Fourier Transform Ion
Cyclotron Resonance (FTICR) has extended the capability of these techniques
for dendrimer analysis [82,100]. Certainly, ESI-FTICR MS was proved useful
in the ionization and characterization of Frechet-type dendrimers with a ben-
zylic hydroxyl end groups [82].
Quadruple ion trap equipped with an ESI was used to investigate the frag-
mentation of PAMAM G0.5 and G1 [86,101]. In both cases, most of the pro-
ducts derived from the precursor ion were associated with those formed via
retro-Michael decompositions reaction. Fragmentation of G1 showed regular-
ity in the product ions and resulted from the loss of 60 Da, obtained by an
intramolecular cyclization, and from the loss of 114 Da, obtained by a four-
centred hydrogen transfer. The fragmentations stemmed either from competi-
tive or from consecutive reactions, even though resonant fragmentation QIT
was used. It was shown in both studies that the principal fragmentation reac-
tion is a retro-Michael rearrangement for G1 and G0.5. Similar study on frag-
mentation of PPI dendrimers G1–G5 was performed by McLuckey et al.
2000, who explained the decomposition by SNi reactions [102].
The type of core dendrimer, nature of the surface groups and number
of generation influence the fragmentation process of the electron-mediated
dissociation, electron capture dissociation (ECD), electron detachment disso-
ciation or collision-induced dissociation (CID) [102]. The investigation of
Kaczorowska and Cooper showed that electron-mediated dissociation of
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 217
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PAMAM dendrimers does not depend on the nature of surface groups, but is
related to the structure of the polymer. It was demonstrated by comparing the
ECD of both PAMAMG1NH2 and PAMAMG2OH, determined by FTICR
MS, which does not affect the ECD fragmentation behaviour. A significant
influence was observed due to the generation number. Both ECD mass spectra
are dominated by fragments that come from the inner generation(s). In con-
trast, CID of the PAMAM dendrimers depended strongly on the nature of sur-
face groups [103].
Another spectroscopic method widely used is NMR, successfully applied
in routine characterization of dendrimers, size and morphology. For dendri-
mers such as PPI, PAMAM, poly(phenyl ester) or poly(ether ketone),1H and 13C NMR are the most used [104–106]. For heteroatom dendrimers
such as phosphorus, carbosilane and silicon-based dendrimers, 31P and 29Si
NMR provides very valuable information. The 31P NMR turned out to be par-
ticularly sensitive to differentiate each layer up to G4 and at least three last
external layers up to G12 [107,108].
UV–Vis and Infra-red methods can be used to monitor the effectiveness of
dendrimer synthesis and to obtain morphological information. IR has been
mainly used to investigate end group transformations, such as the disappear-
ance of nitrile groups in the synthesis of PPI dendrimers [109], the occurrence
of hydrogen bonding in PPI glycine-functionalized dendrimers [110] or the
disappearance of the aldehydes during the synthesis of PMMH dendrimers
[111]. By UV–Vis spectroscopy, due to the fact that the intensity of absorp-
tion band is proportional to the number of chromophoric units, the purity of
dendrimers could be tested [112], but the limit of detection in the case of
PAMAM dendrimer is too high, 10 mg/l [77].
6.2 Scattering Techniques: SAXS, SANS, LLS
The basic element that unites the different scattering mechanisms and is ulti-
mately so important in the study of soft matter is the concept of interference
and its relation to the structure of the soft-matter medium [113]. Small angle
X-ray scattering (SAXS) technique is often used for polymer analysis [114],
and recently for carbosilane and PAMAM dendrimers [115]. SAXS analysis
provides information about the average radius of rotation in solution and the
arrangement of polymer segments. To study the internal structure of dendritic
polymer systems, Prosa and co-workers used SAXS for characterizing the
single-particle scattering factors produced by PAMAM and PPI in solutions
of methanol [116,117]. The SAXS from dendrimers showed similar overall
densities of the dendrimers than those obtained from an electron density mod-
elling. These features were successfully reproduced using a model consisting
of constant-density spheres with a small amount of polydispersity in molecu-
lar size. Scattering from the intermediately sized G4 through G8 dendrimers
reflected a consistent evolution of internal structure progressing from
Analysis and Risk of Nanomaterials218
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‘star-like’ to ‘hard-sphere-like’ organizations [117].The drawback is that it
has a refractive index similar to the dispersant media (water). For this reason,
the intensity of scattering is very low, making it difficult to obtain a consistent
size measurement at an angle of scattering above 100& [118]. This problem
was overcome by Orberg and co-workers using angles of scattering below
100& and with a high concentration of PAMAM particles (18 mg/ml) [119].
They reported size measurements of PAMAM G4 dendrimer in aqueous
media using equipment with a variable angle and a goniometer with an Nd:
YAG solid-state laser-diode light source of wavelength 532 nm. These
measurements closely fit to the size stated by the manufacturer (i.e. 4.5 nm
of diameter at a scattering angle of 90&).
The SANS technique also gives the radius of rotation, but may reveal
more accurate information than SAXS about the internal structure of the den-
drimer and molecular weight; such experiments have been conducted with
PPI, PAMAM [21,120]. A combination of small angle X-ray and neutron-
scattering experiments has been used to study a model system of PAMAM–
copper sulphide nanocomposites in various stages of its formation. They
observed little perturbation of the dendritic species on complexation and a
secondary phenomena of super-molecular aggregation in nanocomposite solu-
tions [121]. Liu and co-workers resolved a controversy between theoretical
and experimental results about the influence of pH on structure of PAMAM
using a molecular dynamic simulation. They discovered small changes in
the size of PAMAM molecule and an internal rearrangement caused by a
pH-induced conformational change from a ‘dense core’ (high pH) to a ‘dense
shell’ (low pH). They suggested how PAMAM dendrimers might be used as
pH-dependent drug delivery systems [120].
6.3 Microscopy: AFM, TEM
The size and shape of dendrimers have been measured using two microscopy
techniques Transmission Electron Microscopy (TEM) and Atomic Force
Microscopy (AFM). TEM requires high vacuum conditions and thin sample
sections to facilitate electron-beam penetration; produces images that can
resolve the order of 0.5 nm. TEM has already proved suitable for imaging
inorganic nanoparticles [122] as well as for the dendrimers G3–G10
having gold covalently attached to each terminal group [123]. AFM has
the advantage of imaging almost any type of surface and produces a high-
resolution and three-dimensional profile. By optical microscopy, visualizing
of PBzE dendrimers with dihydropyrrolopyrroledione core and polyphenylene
dendrimers with perylene imide as end groups was successfully performed
[124]. Other studies on self-assembly of dendrimers have demonstrated its
applicability, for instance, operating in two modes: by tapping for measuring
topographic variations across a surface; and through phase-imaging mode
[125,126].
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 219
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6.4 Chromatographic Separation Techniques: HPLC, UPLC, SEC
LC and size exclusion chromatography (SEC) columns are successfully used
for separation and characterization of different generations of dendrimers.
SEC separate compounds based mostly on the size of a molecule. Its main
advantage is easy handling and high dynamic range concerning molecular
weight determination. SEC separation was performed to determine the abso-
lute weight, intrinsic viscosities and structural characteristics of PAMAM
dendrimer derivatives of different generation number [127,128]. The use of
various SEC columns of different length: 75 mm, 300 mm and various parti-
cle dimensions: 7 mm, 10 mm and 17 mm, showed that SEC is not sensitive
enough to detect and separate impurity species (<10% in the case of single
generation dendrimers) [127].
Both purity and distribution of dendrimers can be analyzed by HPL chro-
matography. Since amine-terminated PAMAM dendrimers adsorb strongly to
a variety of solid media especially hydrophilics, it is recommended to add
counterions or acids to the mobile phase in order to make dendrimer surfaces
hydrophobic (i.e. tetrafluoroacetic acid) [128]. The density of dendrimer–
counterion pairs is a governing factor in the elution of dendrimers through a
hydrophobic column, increasing as a function of the generation number. It
has also been found that, at each generation, there is an overlapping of the
dimer peak with the next generation and the trailing generation with the next
lower generation [77,128,129]. This is consistent with the fact that dendrimers
can contain a small proportion of both trailing generations and dimers result-
ing from synthetic technology [129]. Columns usually used for RP-HPLC in
above-mentioned studies were C5 [77,128,129] and C18 silica based; two
types of mobile phases were used for the elution: water/propanol and water/
acetonitrile both acidified with 0.14% TFA acid or 0.1% formic acid.
Studies comparing HPLC with UPLC showed that the latter technique
gives improvements in efficiency and sensitivity. Results obtained with UPLC
have shown its enhanced potential to assess generational defects and degree of
surface modifications [130]. UPLC analysis of EDA core G4-PAMAM-
(NH2)64 and surface-biotinylated PAMAM G4 produced a sevenfold increase
in average number of theoretical plates, improving the capability to distin-
guish dendrimer surface variances, and reducing the retention time by 36%.
In addition, due to the reduced band spreading during the separation process,
a lower injection volume and concentration of analyte is factible, improving
the limit of detection by a factor of 100 (HPLC method for G4, PAMAMs,
was 1.6'10!10 mol compared to 1.6'10!12 mol for UPLC) [130].
6.5 Electrophoretic Methods
As mentioned before, dendrimers are well soluble in water and may carry
multiple charges. The solubility strongly depends on pH, type of dendrimer
Analysis and Risk of Nanomaterials220
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surface, solvent or temperature. Thus, they can be analyzed by electrophoretic
methods. Electrophoresis is one of preferred separation method in biochemis-
try due to the fact that its equipment is widely available and simple modifica-
tions of analytical conditions allow separating any charged, water-soluble
dendrimers of varied shapes and dimensions (e.g. nucleic acid and polylysine
dendrimers) [131,132].
Sharma and co-workers developed a simple, inexpensive and rapid electro-
phoretic method for routine evaluation of purity for PAMAM dendrimers
G0–G7. PAMAM dendrimer separation was performed under both basic and
acidic conditions, providing an increased resolution and sensitivity under
acidic conditions being PAMAM G0 dendrimer visible at 1.5 mg under these
conditions [132].
A suitable method based on dynamic coating capillary electrophoresis for
the analysis of highly positively charged PAMAM dendrimers has also been
described [133]. The best separation of seven PAMAM generations was
obtained avoiding the interactions between amino and silanol groups using a
dynamic coating-fused silica capillary and a background electrolyte with
tris-phosphate buffer (50 mmol/l, pH 7.4) and 0.05% (w/v) polyethylenei-
mine. Thus, an extremely acidic pH was not necessary for the separation of
these compounds [133].
The influence of cationic PAMAM dendrimers on capillary electroseparation–
UV analysis of proteins has been recently examined. When PAMAM is added to a
buffer, a high selectivity is exhibited towards proteins. PAMAM G1 in 30-mM
phosphate, at pH 2.6 causes formation of protein–PAMAM complex, driven by
hydrophobic attraction, electrostatic interaction and/or hydrogen bonding and
improves the separation of proteins (such as myoglobin and trypsin). Moreover,
those complexes lead to variation in UV absorbance of the proteins, rendering
the possibility of sensitive detection [134].
Some studies have been dedicated to the development of an improved
method combining polyacrylamide gel electrophoresis (PAGE) and capillary
zone electrophoresis (CZE) for the analysis of EDA core PAMAM succi-
namic acid dendrimers (PAMAM-SAHs) and a core–shell tecto (dendrimer)
carrying succinamic acid termini using silanized silica capillary. PAGE
results showed that the relative mobilities of G2–G7 decreased with increas-
ing generation number. Due to the gel filtration effect, native (gradient)
PAGE can separate different PAMAM generations, allowing their purity
and homogeneity to be assessed. Using SDS (sodium dodecyl sulphate)-
PAGE it was possible to estimate the molecular weight of PAMAM-
SAHs based on a protein standard. By a CZE method, PAMAM-SAHs and
a core–shell tecto(dendrimer) were also analyzed. Since all generations
assayed (G1–G7) showed similar electrophoretic mobilities, the polyanionic
PAMAMs are not adsorbed on the surface of the quartz capillary and the sep-
aration is only influenced by the charge/mass ratio and the EOF (electro-
osmotic flow). The movement of ions mainly depended on the electric field
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 221
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and minor differences in charge/mass ratios were insufficient to achieve effec-
tive separations between generations [135,136].
Given the analytical methodologies presented in this section and to our
best knowledge, there are no sensitive and highly selective methods for the
detection of PAMAM (or other type of dendrimers) that reliably detects and
measures such nanoparticles in the environment. The limits of detection
obtained until now by the analytical methods mentioned above leave much
to be desired. The disposal, fate, environmental levels or effects remain unre-
solved, but on the other hand, it is obvious expansion of trade in nanomater-
ials. In the current scenario, new dedicated methods to identify and quantify
nanoparticles in the environment need to be explored.
7. DETERMINATION OF PAMAM DENDRIMERS INNATURAL WATERS BY LC–MS
In this section, we present our approach for the analysis of PAMAM dendri-
mers in natural waters by TOF and QTOF LC–MS systems. Besides the
analytical methodologies previously cited for a characterization in terms of
‘fake defects’, purity or degree of surface functionalization, actual interest is
focussed on making available the detection and measurement of nanoparticles
in complex environmental matrices like natural waters. This may truly be a
challenge, because of the size and small amounts of nanoparticles that make
necessary high-sensitive detection techniques but also extremely selective that
permits their unequivocal recognition with clear analytical signals. A thor-
ough characterization based on physico-chemical properties is also important
to explain behaviour and interactions with environmental constituents of rele-
vant matrices such as soil, sediment or water.
In aqueous media, we have described a characterization study of PAMAM
dendrimer [77]. Size, distribution, morphology, agglomeration state, surface
charge, effects of pH, ionic strength and formation of adducts were the para-
meters evaluated. Of particular interest is the agglomeration phenomenon
which is an essential parameter to properly understanding transport and envi-
ronmental fate and reactivity of nanoparticles. In our previous study, the
agglomeration phenomena of particles were remarkable: (i) due to the interac-
tion with an anionic surfactant (negative charged), such as SDS and, (ii) at pH
higher than 7, due to conformational changes in the ‘dense core’ of dendritic
structure. The techniques used for this characterization study were dynamic
light scattering and high-resolution transmission electron microscopy. How-
ever, while these methods are especially useful to define ‘experimental clean
system’, they do not enable the identification and quantification of nanoparti-
cles in complex matrices.
By LC–MS technique, a method was developed for the analysis of
PAMAM dendrimer in natural waters. The findings from LC analysis demon-
strated that the combination of size-exclusion and reversed-phase columns
Analysis and Risk of Nanomaterials222
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provides the chromatographic separation of four individual generations of
amino-terminated PAMAM dendrimer. Figure 9 shows a chromatogram
obtained in isocratic mode using water with 0.1% formic acid as mobile phase
and where the retention time of the generations G0–G3 increases with
decreasing dendrimer generation.
With the HPLC coupled to a TOF-MS system equipped with an electrospray
interface and operating in positive ionization mode, accurate mass spectra were
recorded across the range 100–2000 m/z. Under these analytical conditions,
the method was solely applicable to the identification of two (G0 and G1) of
the four species considered as low generation of PAMAM dendrimers. Since
amino-terminated PAMAM G2 and G3 have molecular weight of 3252 and
6900 Da, respectively, and the mass range of the TOF-MS was lower, the mass
spectra obtained were not conclusive. For a detailed MS analysis, this was
merely focussed on generation G0 because of its molecular weight (517 Da),
which is within the common mass region of the TOF-MS. The mass spectrum
obtained for G0 exhibited abundant intensity signals for the protonated molecu-
lar ion at m/z 517.3944 and for the sodium adduct, detected at m/z 539.3767.
Mass spectrum of the extracted ion chromatogram (for m/z 517.3915 with a
mass window width of 0.1 Da) also showed a signal at m/z 403.3145 which
can be associated with the loss of an amidoamine group from the PAMAM den-
drimer G0 molecule, in this particular case, PAMAM G0; therefore, it could be
determined with sufficient identification criteria, supported with an average
error in mass accuracy of !3.1 ppm (Figure 10). On the other hand, the weak-
ness of the method was its poor sensitivity since the calculated LOD was
10 mg/l for PAMAM G0 (determined by serial dilution and with a signal/noise
0
0.00
0.05
0.10
0.15
0.20
0.25 G3
G2
G1
G0
0.30
2 4 6 8 10 12 14 16
Time (min)
Absorb
ance (
UA
, l =
210
nm
)
FIGURE 9 HPLC chromatogram of amine-terminated PAMAM dendrimers for the generations
G0–G3 in isocratic mode.
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 223
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+ESI SCAN (8.758 min) Frag=190.0 V PAMAM G0 10 ppm H2O
Total ion chromatogram
Extracted ion chromatogram
PAMAM G0 517.3944
3106
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.0
1 2 3 4 5 6 7 8 9 10
Time (min)
Inte
nsity (
cps)
3105
3.5
3.0
2.5
2.0
1.5
1.0
5.0
0.0
Inte
nsity (
cps)
3104
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
5.0
0.0
Inte
nsity (
cps)
11 12 13 14 15 16 17 18 19 20
1 2 3 4 5 6 7 8 9 10
Time (min)
400120 140160 180200 220240 260 280300 320 340 360380 420 440 460 480 500520 540 560 580600 620640 660 680 700
Mass-to-charge (m/z)
11 12 13 14
539.3767 (1)
[M+Na]+
517.3944 (1)
[M+H]+
403.3145 (1)
425.2963 (1)
15 16 17 18 19 20
A+ESI TIC Scan Frag = 190.0 V PAMAM 10 ppm H
2O
+ESI EIC (514.3000-517.4000) Scan Frag = 190.0 V PAMAM G0 10 ppm H2O
B
C
FIGURE 10 PAMAM G0 dendrimer: (A) Total ion chromatogram (TIC), (B) Extracted
ion chromatogram (XIC) and (C) TOF-MS spectrum obtained from the XIC for its
accurate mass.
Analysis and Risk of Nanomaterials224
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ratio of 3). In addition, it is well known that mass accuracy of TOF analyzers can
be deteriorated by saturation under certain conditions when analyzing samples
at high concentration. As a result, linearity in the analytical signal might be
adversely affected. The calibration curve evaluated over the range of 12–
500 mg/l, had a correlation coefficient (R2) of 0.9939.
To overcome those drawbacks, another step in our work has been to study
PAMAM dendrimers with higher molecular weight (>2000 Da) by the iden-
tification of their multiply charged species produced by using ESI-QTOF-MS
system, in order to gain sensitivity and high selectivity for their detection in
environmental samples.
In brief, in relation to experimental details, the new method by LC–MS
technique, was developed using new generation of TOF and QTOF systems
(TripleTOF 5600 System, AB SCIEX, Concord, ON) connected to an HPLC
with an electrospray interface. The new capabilities combining high resolv-
ing power and mass accuracy with molecular ranges up to 40,000 Da are
unique to get adequate spectra of these compounds. The MS was operated
in full scan TOF-MS mode and the acquisition of accurate mass spectra
was across the range of 100–3500 m/z. This scale allows identification of
amino-terminated PAMAM dendrimers until generation 5. The data
obtained for the MS analysis of PAMAM G2 with molecular weight of
3254 Da, are presented on Figures 11 and 12. Flow injection analysis
300
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
Inte
nsity c
ps
2600
2800
3000
3200
3400
3600
3800
4000
4200
4400
4600
4800
5000466.0480 (7)
466.1911 (7)
*465.9080 (7)
466.3310 (7)
543.5582 (6)
543.7242 (6)
*543.3916 (6)652.06805 (5)
652.2680 (5)
652.4681 (5)
799.8157 (4)
815.3341 (4) 1086.1080 (3) 1108.0912 (3)
1251.3587 (2)1392.4421 (2)
1629.2765 (2)1650.6364 (2)
1651.6400 (2)1108.4239 (3)1086.7738 (3)
815.0841 (4)
814.8331 (4)
407.9206 (8)
408.0456 (8)
408.1705 (8)
*407.7956 (8)
[M + 7H]+7
[M + 8H]+8
[M + 6H]+6
[M + 5H]+5
[M + 4H]+4
[M + 3H]+3 [M + 3Na]+3
[M + 2H]+2[M + 2Na]+2
Spectrum from PamamG G2 + TOF MS (100 - 3500) from 9.188 to 9.304 min,
350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050
Mass/charge (Da)
1100 11501200 125013001350 140014501500 15501600 1650 1700 17501800
FIGURE 11 TOF-MS spectrum of amino-terminated PAMAM G2 dendrimer. Detection of mul-
tiply charged species.
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 225
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Isotopic cluster
(M+7H)+7
Isotopic distribution for(C142H288N58O26 + 7H)+7
Theoretical isotopic ratio
Mass difference DDa = 0.14
DDa
0.14 Da
A Spectrum from Pamam G2 + TOF MS (100 - 3500) from 9.188 to 9.304 min.
466.0480 (7)
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0465.6
464.50
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
465.0 465.5 466.0 466.5 467.0 467.5 468.0 468.5
465.7 465.8 465.9 466.0 466.1 466.2 466.3
Mass/charge (Da)
Mass/charge (Da)
Intensity of monoisotopic peak465.90578–54.48%
Inte
nsity (
cps)
Inte
nsity (
cps)
466.4 466.5 466.6 466.7 466.8 466.9 467.0 467.1 467.2
466.1911 (7)
466.0480 (7)
*465.9080 (7)
466.1911 (7)
466.3310 (7)
466.4710 (7)
466.6142 (7)
466.7543 (7)
466.3310 (7)*465.9080 (7)
466.4710 (7)
466.6142 (7)
466.7543 (7)
Spectrum from Pamam G2 + TOF MS (100 - 3500) from 9.188 to 9.304 min.
Isotopic Distribution for (C142
H288
N58
O28
+ 7H)7+
B
FIGURE 12 (A) Resolved isotopic cluster attributed to [Mþ6H]6þ. Isotopic peak spacing for
the molecular ion cluster of 0.15 Da. (B) Isotopic abundance pattern.
Analysis and Risk of Nanomaterials226
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compound optimization was performed to obtain maximum sensitivity for
identification and detection, by tuning the parameters of declustering poten-
tial (DP), collision energy (CE) and source-dependent parameters. In gen-
eral, the optimum values selected are similar than those used for smaller
molecules, except the (DP) values that are much lower (e.g. 15 V). That
provides a much better signal intensity.
The LC was performed with a reversed phase with C5 analytical column
150 mm length 4.6 mm I.D and 5 mm particle size. Very bad peak shapes
were obtained with C8 or C18 columns. The mobile phase was a combination
of acetonitrile and HPLC-grade water with a flow rate of 0.3 ml/min. The
addition of 0.1% formic acid in both phases was necessary to obtain an
adequate response. The injection volume was 20 ml.
In contrast to the ESI mass spectrum usually observed from small mole-
cules such as the ‘well known’ environmental contaminants, the complexity
of the ESI mass spectrum obtained for PAMAM G2 is much greater. The
MS spectra acquired present multiple mass peaks that make more difficult
an easy interpretation of the accurate mass ions obtained (Figure 11). The
increasing number of surface functional groups, amine groups in PAMAM
dendrimers, drives to the detection of multiply charged species. Further, MS
spectra exhibits ionized species having a single charge, apart from the molec-
ular ion, which might be associated to the loss of functional groups or even to
structural parts of the molecule such as ‘dendrons’.
In a greater depth of the ESI-MS spectrum of PAMAM G2, shows pre-
dominantly, the multiply charged ions corresponding up to charge þ8 at
407.9206. The most abundant cluster of the spectrum was (Mþ7H)þ7 at
m/z 466.0480 followed by to [Mþ6H]6þ at 543.5582, [Mþ4H]4þ at m/z
814.8331, [Mþ5H]5þ at m/z 652.0700. At lower abundance appear sodium
adducts with charge þ3 (Mþ3Na)3þ and þ2 (Mþ2Na)2þ with m/z values
respectively 1108.0912 and 1650.6364. In lower abundance appears peaks
corresponding to the protonation states of þ4, [Mþ4H]4þ at m/z 814.5744
and a sodium adduct at m/z 1650.1292, [Mþ2HþNa]2þ.
The detection of various protonation states is related to the number of
amino terminal groups of the PAMAM molecule, leading to a complemen-
tarity of data referring to the charge state of the molecule of usefullness for
a concise structural assignment. The determination of these multiply
charged species is feasible because of the high mass spectrometric resolu-
tion (up to 30,000 FWHM depending on the m/z selected), which in
turn resolves the separation of the components of each isotopic cluster
corresponding to each charge state. When the resolution is much lower,
such as in quadrupole analyzers, the analysis of multiply charged ions can-
not be performed by separating individual isotopic variants. Thus, for a
charge state n, the detection of the protonation states of the molecule, in
m/z units is 1/n. The mass spectrum obtained, shows the molecular ion,
[MþH]þ at m/z 3255 (but at a very low intensity) and its distincts
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 227
Author's personal copy
protonated species at m/z values corresponding to ([M]7H/7), ([M]8H/8)
([M]6H/6), ([M]5H/5), ([M]4H/4) or the adducts ([M]2Na/2) and ([M]
3Na/3). Likewise, the isotopic peak spacing for the molecular ion cluster
was of 1 Da, and for its protonated species, depending on the charge,
such as for ([M]7H/7), the peaks of the isotopic cluster appear with a mass
difference of 0.14 Da.
The isotopic abundance pattern may serve also for identifying the presence
of dendrimers when conducting analysis of environmental samples. For
instance, the pattern observed for the base peak [Mþ7H]þ7 fits the profile
of the theoretical isotopic ratio generated by computer algorithms for model
recognition technique. That calculates all possible elemental formula for
detected masses using both accurate mass and isotope distribution. As it is
well known, this information is especially useful for detecting characteristic
isotopic distributions (i.e. bromine or chlorine), but also for the most biologi-
cally abundant elements. For molecular masses >2500 Da with high carbon
content, in this case, PAMAM G2 dendrimer, the first peak in the resolved
isotopic cluster representing the monoisotopic mass is not the most abundant
isotopic peak in the spectrum acquired. This is due to the fact that the species
containing one13C atom are the most abundant when the number of carbon
atoms highly increases in a molecule and therefore raises the probability of
containing at least one heavy isotope. Thus, the most abundant isotopic peak
for the cluster [Mþ7H]7þ was at m/z 466.0480 and the monoisotopic mass
was resolved at m/z 465.9080, consistent with the calculated isotopic ratio
which attributes an abundance for both isotopic peak of 100 and 54%, respec-
tively (see Figure 12B).
For an effective identification, mass accuracy was crucial since it provides
more confident ion assignment in recognizing the PAMAM G2 molecule.
Following with the same example, the identification by means the base peak
[Mþ7H]7þ was successful, with an error of !2.1 ppm, between the observed
(m/z at 466.0480) and theoretically most abundant isotopic peak (m/z of
466.0490 provided by the Formula Finder Software, using isotopic distribu-
tion calculator). This deviation is sufficiently small and it may realistically
be attributed to the molecular formula [C142H288N58O28þ7H]7þ from all
the lists of feasible molecular formulas ranked in function of accurate mass
and isotope distribution. Mass measurement errors between the theoretical
and observed most abundant isotopic peaks were in the !2.1 !4.1 ppm range
for the different protonated species detected. In comparison with small mole-
cules, the higher deviation in mass accuracy errors obtained for these large
molecules is attributable to the fact that, as the mass increases, the absolute
mass error in terms of percent or ppm will also increase proportionally (i.e.
0.01% or 100 ppm¼0.1 Da at m/z¼1000, 0.5 Da at m/z¼5000, or 5 Da at
m/z¼50,000). This level of mass accuracy achieved with the QTOF system
can be executed routinely with external calibration (through both an auto-
mated instrument calibration from a reference spray and an auto batch
Analysis and Risk of Nanomaterials228
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calibration during long runs over 24 h) and without having to constantly mon-
itor instrument.
When exploring other signals in the MS spectrum of PAMAM G2 dendri-
mer, a characteristic shape of peak distribution can be observed, which may
help to constraint on its identity. Clearly, the mass spectra acquired in TOF-
MS mode, shows two series of peak distributions attributed to both species
having a single and a double charge, respectively (Figure 11). Particularly,
in the low mass region, prominent groups of signals come up with a peak-
to-peak distance of 44 Da. The second series of equidistant signals with a dis-
tance of 22 Da emerges across the spectra and exhibits a lower intensity. As a
matter of fact, such doubly charged signals might be associated with the series
of mono-charged peaks, given that mass difference (D m/z) is double. These
fragment ions point principally to the loss of ethyl amine groups from the
amidoamine branches of the dendrimer. On the other hand, the presence
of an intense signal at higher masses gives an even more useful fingerprint
characterization of PAMAM G2 dendrimer. An isotopic cluster of the
proton-bound dimer is generated under mild ESI conditions showing at m/z
1629.2765, the most abundant isotopic peak.
Under these conditions, much useful information has been gained for
providing an improved characterization by MS. The identification of
PAMAM G2 dendrimers can be accomplished by high resolution and
mass accuracy making feasible the detection of multiply charged ions, well
resolved isotopic cluster, exploiting isotopic abundance patterns and sub-ppm
accurate mass. Consequently, these more stringent criteria greatly increase the
probability to recognize nanomaterials and its potential applications in envi-
ronmental analysis, in complex matrices. In terms of quantitative analysis,
the results demonstrated an accurate and reproducible quantitation of
PAMAM G2 using the peak base (the most abundant isotopic peak of the
cluster corresponding to [Mþ7H]7þ, at m/z of 466.0480). A linear response
was observed over a range of concentration of 0.5–5 ppm with a correlation
coefficient of 0.9989. The analytical method yields a satisfactory sensitivity
with a LOD of 0.2 mg/l, obtained by direct injection of water samples. That
approach is very promising and it can allow a proper identification and quan-
titation of PAMAM in water samples.
ACKNOWLEDGEMENTS
We thank the financial support of the Spanish Ministry of Education and Science for the
Project ‘NANOQUAL, Nanoparticles and Water Quality’ (National Plan for Scientific
Research, Development and Technological Innovation, 2008–2011). M. M. U. acknowl-
edges the research fellowship from the Marie Curie Actions (FP7). The authors thank
Dr. Jianru Stahl-Zeng from AB Sciex for technical support. A. U. acknowledges the research
fellowship from the Spanish Ministry of Science and Innovation, project NANOAQUAL
(Ref. CTM2008-04239).
Chapter 6 Chemical and Ecotoxicological Characterization of Dendrimers 229
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