Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting

Article abstract of DOI:10.1016/j.ejmech.2011.11.027

Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This approach is typically achieved by the functionalization of a nanoparticle with two distinct types of molecules, a targeting ligand specific for a cancer cel

Full text of DOI:10.1016/j.ejmech.2011.11.027

European Journal of Medicinal Chemistry 47 (2012) 560e572
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Dendrimer-based multivalent methotrexates as dual acting nanoconjugates
for cancer cell targeting
,
,
a
a
a,c
Ming-Hsin Li a b, Seok Ki Choi a , Thommey P. Thomas , Ankur Desai , Kyung-Hoon Lee
,
*
Alina Kotlyar a, Mark M. Banaszak Holl a
d, James R. Baker Jr. a
,
b
,
c
,
,b,
**
a Michigan Nanotechnology Institute for Medicine and Biological Sciences, and Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
b Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
c Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
d Department of Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This
approach is typically achieved by the functionalization of a nanoparticle with two distinct types of
molecules, a targeting ligand specific for a cancer cell, and a cytotoxic molecule to kill the cell. The
present study aims to evaluate the validity of an alternative simplified approach in the design of cancer-
targeting nanotherapeutics: conjugating a single type of molecule with dual activities to nanoparticles,
instead of coupling a pair of orthogonal molecules. Herein we investigate whether this strategy can be
validated by its application to methotrexate, a dual-acting small molecule that shows cytotoxicity
because of its potent inhibitory activity against dihydrofolate reductase and that binds folic acid receptor,
a tumor biomarker frequently upregulated on the cancer cell surface. This article describes a series of
dendrimer conjugates derived from a generation 5 polyamidoamine (G5 PAMAM) presenting a multi-
valent array of methotrexate and also demonstrates their dual biological activities by surface plasmon
resonance spectroscopy, a cell-free enzyme assay, and cell-based experiments with KB cancer cells.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 8 September 2011
Received in revised form
8 November 2011
Accepted 15 November 2011
Available online 23 November 2011
Keywords:
Folate receptor
Methotrexate
Poly(amidoamine) dendrimer
Targeted drug delivery
Multivalent binding
Dual activity
1. Introduction
Applications of multifunctional NPs in anticancer therapeutic
delivery have been well demonstrated by use of targeting ligands
Nanotechnology is uniquely suited for providing multifunc-
tional platforms for targeted delivery in several life-threatening
diseases, including cancers and inflammatory diseases [1e6]. This
study aims to investigate a novel and simplified delivery strategy
based on functionalization of a nanoplatform with a dual-acting
small molecule, in lieu of two single-acting molecules, that serves
as both a targeting ligand and an anticancer therapeutic. In this
communication, we demonstrate the validity and effectiveness of
this simplified strategy by designing multivalent NPs presenting
methotrexate on the periphery of a generation 5 polyamidoamine
(G5 PAMAM) dendrimer.
specific for a cell surface molecule overexpressed in cancer cells,
such as folic acid receptor (FAR) [7e9], riboflavin receptor [10,11],
avb3 integrin [12e14], prostate-specific membrane antigen [15],
Her2 [16], transferrin receptor [17], and epidermal growth factor
receptor [16,18,19]. The biological process for the effective uptake of
such NPs requires multiple-ligand molecules attached to the NP
surface. It begins with specific adhesion of an NP to the targeted cell
surface in a mechanism that is characterized by multivalent inter-
actions occurring collectively at the interface of multiple receptor-
ligand pairs [12,20]. Such a multivalent mechanism is considered as
highly important during the receptor-mediated endocytosis
because it constitutes the basis for tight NP-cell adhesion and
conformal contacts created during the formation of coated pits
[21e24]. Therefore, in a rational design for targeted NPs, each NP is
covalently conjugated with multiple copies of a targeting ligand on
its periphery in order to achieve the multivalent effects, and each is
further functionalized to carry therapeutic or imaging molecules as
the payloads for cellular delivery [1,25e27].
Abbreviations: FA, folic acid; FAR, folic acid receptor; MTX, methotrexate;
PAMAM, polyamidoamine; SPR, surface plasmon resonance; DHFR, dihydrofolate
reductase.
* Corresponding author. Tel.: þ1 734 615 0618; fax: þ1 734 615 0621.
** Corresponding author. Michigan Nanotechnology Institute for Medicine and
Biological Sciences, and Department of Internal Medicine, University of Michigan,
Ann Arbor, MI 48109, USA. Tel.: þ1 734 647 2777; fax: þ1 734 615 2506.
E-mail addresses: skchoi@umich.edu (S.K. Choi), jbakerjr@umich.edu (J.R. Baker).
Despite the rational basis of the NP design and successful proof
of concept studies demonstrated already, several challenging issues
face the development of cancer-targeting therapeutic NPs. They are
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.027

M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
561
attributable simply to the complexity of the NP structure and the
lack of methods to control the distribution of the particle size,
ligand density, and drug loads [27e31]. Currently, there are only
a few specialized methods demonstrated for the precise engi-
neering and ligand functionalization of NPs such as PAMAM den-
drimer [28,30,32], polymer [33], or gold [34,35]. To ease the
complexity of the fabrication of targeted NP therapeutics, we have
explored new design strategies. Here we evaluate the feasibility of
using a dual-acting small molecule that can: i) function as a ligand
for a cancer-specific receptor, and ii) induce cytotoxicity following
cellular internalization. Because this approach is based on using
a single type of small molecule for both targeting and functional
activity, the precision by which these functionalized nanoparticles
can be synthesized is higher than what can be obtained using the
conventional two small molecule approach.
In our search for candidate molecules that could both target and
function as a therapeutic, we were interested in methotrexate
(MTX, Fig. 1), which has been used as an anticancer drug [5,6]. This
therapeutic molecule functions primarily by inhibiting the meta-
bolic enzymes human dihydrofolate reductase (DHFR), an enzyme
localized in the cytoplasm (Ki ¼ 1.2 nM) [36]. While the cellular
uptake of MTX is mediated by reduced folate carrier proteins [37],
MTX is also able to bind FAR because of its high structural
homology to FA, though at
a
lower affinity constant
(KD ¼ w20e100 nM vs. KD (FA) ¼ w1 nM to kidney FAR) [38e40].
Despite the lower FAR affinity of MTX, we hypothesize that MTX
could still function as a targeting ligand if multiple copies per
nanoparticle were utilized to form a multivalent nanoparticle
[21e24]. Recently, we designed a class of synthetic multivalent
nanoparticles, each composed of multiple FA ligands conjugated to
a G5 PAMAM dendrimer scaffold, and demonstrated by surface
plasmon resonance (SPR) spectroscopy that nanoparticles func-
tionalized with more than one FA bound more tightly to a bovine
folate binding protein (FBP)-coated surface than free FA [20,41].
Based on this observation, we aimed to determine whether
a multivalent MTX-based dendrimer is still effective for FAR tar-
geting. A recent theoretical analysis suggests that multivalent
cooperativity can be kinetically limited if the binding of an indi-
vidual receptor-ligand pair is too tight [42]. It further suggests that
the targeting specificity for a particular cell type can be enhanced
by making the affinity of each individual receptor-ligand pair
weaker. In this study, we describe the synthesis of multivalent MTX
conjugates and provide evidence, based on SPR and in vitro studies,
that these conjugates possess the dual activity necessary for
serving as an effective cancer therapeutic.
a
b
2. Results and discussion
2.1. Surface plasmon resonance (SPR) spectroscopy of multivalent
MTX conjugates
We studied the effect of multivalency on the interaction between
FBP present on the surface and a dendrimer functionalized with
MTX molecules by using SPR spectroscopy. As a bioanalytical
c
Fig. 1. (a) Structure of folic acid (FA), and methotrexate (MTX); (b) Schematic for the folic
acid receptor (FAR)-mediated cancer cell targeting by a dendrimer nanoconjugate
(G5-FAn-MTXm) presenting FA as a targeting ligand and carrying MTX as a cytotoxic drug;
(c) Schematic for the proposed folic acid receptor (FAR)-mediated cancer cell targeting by
a dendrimer nanoconjugate (G5-MTXn) presenting methotrexate (MTX) as a dual-acting
molecule for targeting and the cytotoxic activity. The figure is not drawn to scale.
Scheme 1. A representative synthetic scheme for G5 PAMAM dendrimers presenting
either folic acid (FA) or methotrexate (MTX) molecule in multiple copies. (i) Ac2O,
MeOH, rt; (ii) folic acid, EDC, DMSO, DMF, rt, 24 h; (iii) Ac2O, DMSO, rt; (iv) variable
amounts of methotrexate, EDC, DMF, DMSO, rt, 24 h.

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M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
Fig. 2. Surface plasmon resonance (SPR) experiments measuring the binding (association, and dissociation) kinetics of FA, MTX, and an MTX-presenting dendrimer 2c to bovine
folate binding protein (FBP) immobilized onto a CM5 sensor chip. (a, b) SPR sensorgrams for monovalent ligands FA, and MTX, respectively, each injected at three different
concentrations. (c) SPR sensorgrams for Ac-G5-(MTX)5.0 2c, injected at 2.0
mM (an upper curve), and 1.3 mM (a lower curve). (d) An illustration for fraction of dendrimer 2c desorbed
at the dissociation phase (%decrease in RU), each corresponding to the mean value calculated from the two SPR sensorgrams (c).
D
method well established for studying binding kinetics of analytes to
the biological surface on a real-time basis, SPR spectroscopy has
been utilized for the studies of multivalent ligandereceptor inter-
actions, including the system of the G5 PAMAM dendrimer conju-
gated with FA [20,43e47].
2.1.2. Surface plasmon resonance (SPR) spectroscopy
For the SPR experiments we utilized a CM5 sensor chip to
present bovine FBP immobilized on the surface. Following an EDC-
based amide coupling method as described previously [20], the
FBP-presenting chip was prepared at the surface protein density of
9.5 ng/mm2 (z2 FBP per 10 nm2). It was first used for the binding
studies of the monovalent control ligands (FA, MTX) and a set of
their SPR sensorgrams are shown in Fig. 2a,b. Each of the sensor-
grams for the monovalent ligands was analyzed to extract its
kinetic parameters (on rate ¼ kon, off rate ¼ koff) by using the
BIAevaluation software, version 3 (Biacore). Given the monovalent
mode of association for free FA or MTX, a 1:1 binding mode was
utilized for a curve fitting based on the Langmuir model [20], which
led to determination of the kinetic parameters (koff, kon) and as
2.1.1. Synthesis of dendrimer-based multivalent MTX conjugates
In designing multivalent MTX conjugates we followed the
approach previously developed for the SPR studies of dendrimer-FA
conjugates [20] (1, Scheme 1) because of the similarity in the
structure and chemical reactivity of FA and MTX. Scheme 1 shows
the synthetic method and structures for multivalent MTX conju-
gates 2aed studied here. Each of the conjugates contains a variable
number of MTX molecules presented on the neutral surface of G5
PAMAM dendrimer. Synthesis of these conjugates was performed
by using an EDC-based coupling method where an amide bond is
a result, the equilibrium dissociation constant (KD ¼ koff/kon
;
Table 1). The results suggest that MTX binds to bovine FBP with
a high micromolar dissociation constant (KD ¼ 2.4 ꢀ 10ꢁ5 M), an
affinity w2-fold lower than that of FA (KD ¼ 1.1 (ꢂ1.0) ꢀ 10ꢁ5 M in
this study; 5 (ꢂ3) ꢀ 10ꢁ6 M [20]), primarily as a result of its slightly
higher off rate.
formed between the carboxylic acid of an MTX L-glutamate and
a primary amine present on the periphery of a partially acetylated
dendrimer molecule [20]. Following the purification using exten-
sive dialysis (MWCO 10 kDa) first against phosphate-buffered
saline (PBS) solution and then deionized water, each of these
conjugates was characterized in combination with MALDI-TOF
mass spectrometry, 1H NMR spectroscopy, and UV/Vis spectrom-
etry. These analyses allowed us to calculate the number of ligand
molecules attached per dendrimer, though on a mean basis: 1
(Ac-G5-FA8.2), and 2aed (Ac-G5-MTXn; n ¼ 1.1, 2.6, 5.0, 7.1).
Binding studies of dendrimer-based multivalent MTX conju-
gates 2aed were then performed using the same FBP-presenting
CM5 chip. SPR sensorgrams for the conjugates to the FBP surface
are illustrated in Fig. 2c by the conjugate 2c where each set of the
sensorgrams shows the association and dissociation curve. PAMAM
dendrimer negative controls, conjugated without either MTX or FA,
Table 1
Rate constants and equilibrium dissociation constants (KD) of folic acid (FA), methotrexate (MTX), Ac-G5-(FA)8 (1), and Ac-G5-(MTX)5 (2c) to the folate binding protein
measured by surface plasmon resonance spectroscopy.
Ligands
FA
MTX
1
2C
KD (M)a
1.1 (ꢂ0.50) ꢀ 10ꢁ5
1.1 (ꢂ1.0) ꢀ 103
1.2 (ꢂ0.2) ꢀ 10ꢁ2
1
2.4 (ꢂ0.11) ꢀ 10ꢁ5
7.0 (ꢂ5.6) ꢀ 102
1.7 (ꢂ0.6) ꢀ 10ꢁ2
1
2.3 (ꢂ0.35) ꢀ 10ꢁ9
2.9 (ꢂ2.2) ꢀ 105
6.6 (ꢂ0.76) ꢀ 10ꢁ4
4820 (588)c
2.6 (ꢂ1.0) ꢀ 10ꢁ8
3.4 (ꢂ2.5) ꢀ 104
8.7 (ꢂ2.5) ꢀ 10ꢁ4
923 (185)c
kon (Mꢁ1sꢁ1
)
koff (sꢁ1
)
bb
a
Each dissociation constant (KD ¼ koff/kon) represents a mean value calculated by averaging the data obtained from at least three sets of measurement per injection
concentration.
b
c
b
¼ multivalent binding enhancement ¼ [KmD ono O KDmulti].
Valency (n)-corrected value ¼ [
b O n] where n is equal to either 8.2 (1) or 5.0 (2c).

M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
563
were also prepared for binding and include a fully acetylated
neutral dendrimer (Ac-G5) and a glutaric acid-terminated den-
drimer (G5-glutaric acid). Neither of these dendrimers showed any
meaningful level of specific adsorption to the FBP surface when
measured under comparable conditions, indicating the specificity
of the FBP binding (Figure S8). As an evidence indicative of tight
adsorption, the SPR profiles of 2c are characterized markedly by
slow dissociation (Fig. 2c,d), a kinetic feature suggesting the
multivalent binding as consistently observed in numerous other
multivalent systems [20,43,44]. Such a binding feature was also
observed similarly as the binding of Ac-G5-FA8.2, a multivalent
dendrimer comparator presenting folate ligand as a positive control
(Table 1; Figure S8) [20]. The dissociation curves for 2c suggest that
it dissociates apparently in multiple phases, initially at a rapid off
rate (¼
D(DRU)ODt) and subsequently at slower off rates. For
example, w35% fraction of desorption was observed per initial 20 s,
followed by w18% of fractional desorption per following 20e200 s
until the end of data collection time (200 s), when the dissociation
appears to still be incomplete (Fig. 2d).
2.1.3. Correlation between dendrimer population and fractional
desorption
We hypothesize that such mixed dissociations observed from
2c could reflect the distribution of its subpopulations on the
assumption that some subpopulations bind weakly and others
bind more tightly and dissociate more slowly. The possibility of
such mixed dissociations might be accounted for by the preex-
isting heterogeneity in the dendrimer population of 2c, in partic-
ular in regard to the distribution in ligand valency. While 2c Ac-
G5-MTX5 is assigned to have five MTX ligands attached per den-
drimer as calculated on an average basis, our recent studies
[28,32] indicate that it is composed of diverse multivalent den-
drimer species that can be grouped by their ligand density
according to Poisson distribution (Fig. 3a). Of those mixed den-
drimers having variable ligand valency, we believe that the lower-
valent species are likely to bind less tightly than the higher-valent
species if predicted on the basis of the large number of related
studies [20e23]. We address this hypothesis in Fig. 3b, where the
fractional desorption of G5-MTXn 2ae2d is plotted as a function of
the mean valency of the MTX for each conjugate. The plot shows
a positive correlation for the three conjugates except for 2d, which
showed otherwise deviation, perhaps due to its partial solubility,
and suggests that the observed degree of desorption is greater for
those conjugates having a lower MTX valency. Fig. 3b also shows
Fig. 3. (a) Poisson distribution of dendrimer-methotrexate conjugates G5-MTXn
2ae2d, each having the MTX mean of 1.1, 2.6, 5.0, or 7.1, respectively. (b) A plot of
fractional desorption of G5-MTXn 2ae2d as a function of mean MTX number (valency).
The fractional desorption was calculated from the SPR binding studies of the den-
P
i
drimer conjugates. The sum of the fractions
(
n¼0Frn) for multivalent species
(G5-MTXn; n ¼ 0e18) distributed in each of the dendrimer-MTX conjugates is also
plotted separately in the secondary y-axis. The value for Frn is defined as the fraction of
n-valent species relative to all the species that comprise each conjugate. As an illus-
P
i
tration, each value for n¼1Frn (where i ¼ 2) in 2ae2d refers to the sum of the two
fractions for a monovalent (Fr1) and divalent (Fr2) species, each calculated by Poisson
distribution of the conjugates above.
employed for affinity analysis based on either a monovalent or
bivalent binding mode and is also used for studies to determine
reasonable estimates of the avidity constants for multivalent ligands
[43,46,48e50], including those that are nanoparticle-based
[20,47,51]. Application of these analyses for the sensorgrams of
P
i
fractions ð n¼0FrnÞ for major low-valent species (n ¼ 1e4) that
are distributed in each of the conjugates, as calculated on the basis
of their Poisson distribution (Fig. 3a). It suggests that a fraction of
dendrimer desorption could be accounted for further by the
fraction of low-valent speciesdexcept for 2d. Specifically, the sum
of fractions from monovalent to tri- or tetravalent species
P
i
(
n¼0Frn; i ¼ 3, or 4) is best correlated with the fraction of
desorption within an experimental error range. A recent work by
Waddell et al. [41] hypothesizes that all desorption observed in
the analogous G5-FA system is due to the dendrimer with a single
ligand attached. This G5-FA work conforms to the i ¼ 1 data (Fr1)
in Fig. 3b. Although the trend is similar in our work, greater
agreement has been found for i ¼ 3, where the desorption is more
correlated with the fractions of dendrimers conjugated with up to
3 ligands.
2.1.4. Determination of equilibrium dissociation constant (KD)
In an approach to better understand the complicated dissociation
profiles for 2aed, we estimate their dissociation constants by using
global fittings (BIAevaluation software) that include Langmuir and
bivalent analyte models. Each of these models is frequently
Fig. 4. A summary of equilibrium dissociation constants (KD, M) for dendrimer-based
multivalent ligands (1, 2aed) plotted against ligand valency (n). Dissociation constants
for control ligands including folic acid (FA) and methotrexate (MTX) are placed arbi-
trarily around n z 0 as the reference values indicative of monovalent association.

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M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
Ac-G5-(MTX)5 2aed allowed us to extract the estimates for their
kinetic rate constants (kon, koff) and, consequently, for the steady
state dissociation constants (KD) for the folate binding protein
immobilized on the surface, as summarized in Table 1 and Fig. 4 (see
also Table S3). As an illustration, the dissociation constant estimated
for 2c (KD ¼ 2.6 ꢀ 10ꢁ8 M) suggests that its binding avidity is
enhanced by a factor of w900 (multivalent binding enhance-
ment ¼
b
¼ [KDmonoOKDmulti]) relative to free MTX (KD ¼ 2.4 ꢀ10ꢁ5 M).
a
b
Scheme 2. (a) Synthesis of a new methotrexate-linker construct. Reagents and conditions: i) bromoacetyl chloride, DIPEA, CHCl3, 0 ꢃC to rt, 24 h, 64%; ii) methotrexate hydrate,
Cs2CO3, K2CO3, NaI, DMF, rt, 48 h, 22%; iii) TFA, CH2Cl2, rt, 15 min; (b) Synthesis of generation 5 (G5) PAMAM dendrimer-based nanoconjugates 7a,b, 8a-d, and 9a,b with variable
amounts of methotrexate attached singly or in combination with folic acid molecules. iv) EDC, NHS, DMF, rt, 36 h; v) 5, Et3N, DMF, rt, 24 h; then ethanolamine;
vi) 5, FA-CONH(CH2)2NH2, Et3N, DMF, rt, 24 h; ethanolamine; vii) 5, fluorescein-NHC(]S)NH(CH2)4NH2, Et3N, DMF, rt, 24 h; ethanolamine.

M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
565
Except for 2a (Ac-G5-MTXn: n ¼ 1.1), which bound weakly, like
a monovalent ligand, the other conjugates 2b and 2d showed the
binding kinetics characterized by slow off rates (Table S3) with their
dissociation constants (KD) lowered by two to three orders of
magnitude relative to that of MTX (Fig. 4). We believe that this
observation is consistent with the hypothetical mechanism for
multivalent association, where complete dissociation by a multiva-
lent ligand occurs much more slowly because all of the ligands
tethered to a single multivalent particle have to dissociate simul-
taneously from multiple receptor sites (observed off rate ¼ koff,
n
z kd where kd refers to the mean rate of dissociation for
obsd
a single receptor-ligand pair and n refers to the multivalency in
bound states) [23,43,44]. In summary, the SPR study demonstrates
that the dendrimer-based multivalent MTX conjugate binds much
more tightly to the multivalent FAR surface than free MTX. This
SPR study provides evidence that FAR could be targeted by the
MTX-based multivalent platform.
2.2. Design and synthesis of PAMAM dendrimers conjugated with
MTX through a longer linker framework
After performing SPR binding studies at the receptor level, we
proceeded to the biological assay at the cellular level for the den-
drimers conjugated with MTX 2aed to determine if these conju-
gates are biologically active in inhibiting cell growth. In a cell-based
XTT assay using KB cancer cells, a sub-line of cervical carcinoma
that expresses a high level of folate receptor [52], all of these MTX
conjugates were weakly cytotoxic at low micromolar concentra-
tions (<10% inhibition of cell growth, data not shown). We
hypothesized that the weak cytotoxicity might be attributable in
part to the direct amide linkage used in 2aed because this linker
could be too stable for rapid release of the MTX, following the
cellular uptake of the conjugates. In order to optimize the biological
activity by the MTX conjugates at the cellular level, we designed
a new MTX linker construct 5, made through two major modifi-
cations: i) the functional group at the linkage point that affects its
chemical stability; ii) the tether length (Scheme 2). In this linker
construct, we introduced a less stable ester linkage placed at the
g-carboxylate oxygen of the MTX L-glutamate and, in combination,
a longer linker that consists of a 6-atom spacer terminated with
a primary amine, the functional group introduced for conjugation
with glutaric acid-terminated PAMAM dendrimer [53] later. The
ester linkage is more likely hydrolyzed inside the cell by the actions
of intracellular esterases and/or in acidic endosomes (pH z 5e6.5)
where FAR-associated conjugates are thought to be localized
following cellular entry [54,55]. Therefore, under intracellular
conditions, the ester linkage may be cleaved for drug release in
a rate much faster than the amide linkage used in dendrimer
conjugates 2. The longer spacer for MTX tethering, which is esti-
mated to be 14.8 Å in distance from the dendrimer surface when
calculated at a fully extended conformation (ChemBio3D Ultra
12.0), can provide the tethered MTX with more conformational
flexibility of the linker and freedom of movement. Such a longer
tether might play a critical role in the interaction at the enzy-
meedendrimer interface when the tethered MTX molecule inhibits
the catalytic activity of DHFR (cf. the crystal structure of hDHFR [56]
in complex with a methotrexate molecule at its active site in Fig. 5)
[16,57].
Fig. 5. (a, b) molecular models generated by molecular dynamics simulation for the
representative members of generation 5 PAMAM dendrimer conjugated with metho-
trexate, either directly via an amide bond (2c, Scheme 1) or through a medium length
of spacer (7a, Scheme 2). Distribution of MTX molecules on each of the dendrimers
was selected arbitrarily for this simulation purpose, and thus should be considered as
one of the potential distributions. Final configurations for 2c (a) and 7a (b) were
acquired after 1-ns MD simulations. The figures of the structures were generated with
the software VMD (visual molecular dynamics) where the PAMAM dendrimer and
MTX were shown as lines and van der Waals surface, respectively (Colors: grey:
Dendrimer, light blue: carbon, blue: nitrogen, red: oxygen); (c) A crystal structure of
human dihydrofolate reductase (hDHFR) in complex with a methotrexate molecule at
its active site (PDB code 1u72), where the L-glutamate carboxylic acids from the drug
molecule are anchored near the entrance to the enzyme catalytic pocket, while
a pteridine head group (hidden) is bound deep into the pocket. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)

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M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
2.2.1. Molecular dynamics (MD) simulation of dendrimer-MTX
conjugates
2.2.2. Synthesis of dendrimer conjugated with MTX via longer
linker
In an effort to corroborate this hypothesis for the structural
features of the PAMAM dendrimers and the role played by the
linker, we constructed molecular models for the two representative
members of MTX-conjugated PAMAM dendrimers, each conjugated
either directly via an amide linkage (2c, Scheme 1) or through the
longer spacer (7a, the dendrimer conjugated with the longer linker
construct 5, Scheme 2). Fig. 5 shows these two models generated by
Molecular Dynamics (MD) simulations based on an implicit solvent
model and using the distribution of MTX molecules that were
selected arbitrarily for the simulation purpose [58,59]. The radius of
gyration estimated for each of the dendrimers, 2c and 7a, is
20.6 ꢂ 0.2 and 22.0 ꢂ 0.2 (Å), respectively. The model for 7a
suggests that the longer linker is flexible in the conformation and
can provide a variable distance for the spacer from the dendrimer
surface. Accordingly, it can provide each of the attached MTX
molecules with a greater level of exposure to the solvent.
MTX linker 5 was then prepared using a two-step process based
on the synthesisof 4 bythe O-alkylationof the MTX
a bifunctional linker 3 (Scheme 2a; regioselective ratio for
-ester ¼ 10.2 after purification). Subsequent deprotection of the
L-glutamatewith
g-ester to
a
N-Boc group with the TFA led to 5 isolated as a TFA salt. Synthesis of
G5 PAMAM dendrimers conjugated with the new MTX linker
construct (7a,b, 8aed, and 9a,b) was performed by the covalent
attachment of 5 to the G5eCO2H 6 (Mn ¼ 42730 gmolꢁ1, PDI ¼ Mw/
Mn w 1.046) [53] through an amide linkage (Scheme 2b). Members
of the conjugates include 8aed, which contain MTX as well as FA
molecules co-attached as the targeting ligand. Other dendrimer
conjugates include 9a,b, each containing FITC as a fluorescent
imaging molecule. Each of the mentioned dendrimers was synthe-
sized by a one-pot reaction process in which the carboxylic acids
located at the periphery of the PAMAM dendrimer (w100 CO2H per
dendrimer molecule) were preactivated to N-hydroxysuccinimide
Fig. 6. (a,b) Dose-dependent binding and uptake of PAMAM dendrimer conjugates 9a,b in KB cells. The cells were incubated with each of the conjugates at different concentrations
for 2 h, rinsed and measured for its mean fluorescence in a flow cytometer. For competitive ligand displacement experiments, the cells were treated with the conjugates under the
condition identical to the above except in the presence of free folic acid (50 mM); (c) Confocal microscopy of KB cells treated with 9b (G5-MTX7.5-FITC2.4; 100 nM). KB cells were
incubated with the indicated conjugate for 18 h, fixed and treated with a nuclear staining agent, DAPI (40,6-diamidino-2-phenylindole) prior to the measurement of DAPI (blue) and
FITC (green) fluorescence using a confocal microscope. Control experiments were performed under an identical condition using G5-FITC1.4 (300 nM) as a non-targeting dendrimer
conjugate, and PBS alone. An optical microscopic image for each confocal image is shown in the upper part. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
567
esters and coupled with 5, singly or in combination with FA-
CONH(CH2)2NH2 [60] and/or fluorescein isothiocyanate (FITC)-dia-
minobutane as an imaging molecule [61]. Each of the resultant
dendrimer conjugates was purified by extensive dialysis using
membrane tubing (MWCO 10 kDa) against phosphate-buffered
saline (PBS) solution and then water and was characterized by
analytical methods including MALDI-TOF mass spectrometry, 1H
NMR spectroscopy, UV/Vis spectrometry, and analytical HPLC
(purity >96%) developed in our laboratories. Analysis of these data
allowed us to calculate the number of FA, MTX, and imaging mole-
cules attached perdendrimer particle on a mean basis (Table S2). The
molecular masses for these dendrimer conjugates are in the range of
45,000e48,000 (gmolꢁ1): 7a ¼ 45,700, 7b ¼ 47,200 (PDI ¼ 1.103 by
gel permeation chromatography), 8a ¼ 46,700, 8b ¼ 47,200,
8c ¼ 47,800, 8d ¼ 48,100, 9a ¼ 46700, 9b ¼ 48,350.
2.3. Cell-based experiments in KB cancer cells
First, we performed cellular binding and uptake in KB cells that
express a high level of folate receptor (FAR) [52], using fluorescent
dendrimer conjugates 9a and b, each presenting MTX alone
without FA attached. Fig. 6 shows that each of these conjugates
bound to the cells in a dose-dependent manner at concentrations
up to 1000 nM. In addition, under this binding condition, consid-
ered identical or comparable, 9b (7.5 MTX per dendrimer) showed
a slightly greater level of cellular association than 9a (5 MTX per
dendrimer). The binding activity of each of the MTX conjugates
could be blocked completely when each conjugate was co-
incubated with free FA dosed at a higher concentration (50 mM).
However, it is notable that the dendrimers conjugated with MTX
alone did not show concentration-dependent saturation curves,
a phenomenon which is often displayed by FA-presenting den-
drimer conjugates under comparable assay conditions [26,52,62]. It
is possible that the lack of such binding saturation might be
attributable to a lower avidity binding in combination with a slower
rate of cell uptake, compared to FA-mediated cell targeting. The
uptake of the conjugate 9b in KB cells was then studied by confocal
microscopy, as illustrated in Fig. 6c in which the green fluorescence
in the cytoplasm indicates that the dendrimer particles were
internalized. The results from the cell binding studies suggest that
FAR molecules are involved in the surface receptor targeted by
these MTX conjugates.
Second, we evaluated the cytotoxicity of the dendrimer-MTX
conjugates 7a, 7b, 8aed, 9a, and 9b, each conjugated with the
new MTX linker, against FAR-overexpressing KB cells in an XTT
assay [29]. As shown in Fig. 7a, the dendrimer conjugates 7a and 9a,
each presenting MTX molecules alone even without FA, potently
inhibited cell growth in a dose-dependent manner in which
a maximal inhibition (w50% inhibition of cell growth) was ach-
ieved, each at w25 nM. In contrast, negative controls (such as
a fully acetylated PAMAM dendrimer, and other dendrimers
conjugated with FA and/or dye alone) did not show any cytotoxicity
to KB cells under an identical assay condition (not shown) [10,52].
Each of the conjugates 7a, 9a was more cytotoxic than 10 (G5-FA5-
MTX7.5), the internal standard having FA ligands co-attached
directly through the amide bond [1,52]. This positive control is
structurally analogous to 1 (Scheme 1) but contains fewer FA
(5 copies per dendrimer) as well as MTX (7.5 per dendrimer)
molecules which are attached in an ester linkage [29,52]. Fig. 7a
also shows cytotoxic activities for other analogous MTX conjugates,
8a and 8b, as the positive controls, each targeted by co-attached FA
ligands. Co-presentation of FA in each of the comparators led to
a slight increase in the inhibition activity, possibly as a result of the
targeting effect contributed by FA ligands during the cellular
uptake. The other series of dendrimer-MTX conjugatesd7b, 8c, 8d,
Fig. 7. (a,b) Cytotoxicity of G5 PAMAM conjugates 7a,b, 8aed, 9a,b, and 10 in FAR-over
expressing KB cells. KB cells were incubated with each of the dendrimer conjugates for
2 d, and allowed to grow further for 2 d. The level of cytotoxicity was quantified by
a
colorimetric assay based on XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4-
tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate); (c) Inhibition of
human dihydrofolate reductase (hDHFR) by 7b (G5-MTX7.5) conducted in a cell-free
enzyme assay. The unit for DHFR activity is defined as
concentrations for 7b on the X-axis are given on the basis of MTX rather than den-
m
mol ꢀ minꢁ1 ꢀ mgꢁ1, and
drimer conjugate.
and 9bdeach having a higher number of MTX molecules (7.5)
carried per dendrimer, also showed potent cytotoxicity against the
FAR-overexpressing KB cells. These conjugates were generally more
active than those comparable conjugates otherwise having a lower
number of MTX molecules (5) (Fig. 7b). While it is consistently
observed that presentation of MTX alone in the conjugates 7b and
9b was sufficient to induce cytotoxicity, the MTX conjugates 8c and
8d that present FA as well were more active than those without co-
attached FA. Also, one of the conjugates, 8d, was more potent than
free MTX, an activity which is rarely observed among the standards
of our previous results from other FA-targeted MTX conjugates
[29,52]. In summary, the cell assay demonstrated that PAMAM
dendrimers conjugated with MTX alone could cause potent

568
M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
cytotoxicity in KB cells with the IC50 values of 25 (7a, G5-MTX5
series) to 10 nM (7b, G5-MTX7.5 series) calculated on a conjugate
basis. The co-attachment of FA as a targeting ligand led to a synergy
in the biological activity and led to greater cytotoxicity. This
suggests that an MTX molecule tethered to a dendrimer nano-
particle is both a ligand required for targeting a FAR-expressing cell
and a drug molecule that kills the cancer cell. The level of cellular
activity appeared to be tunable in response to the variation of the
MTX valency and the co-presentation with FA ligands.
4. Experimental section
4.1. General
General synthetic methods, and the details of instrumental
analysis for the characterization of small molecules and dendrimers
by using 1H, and 13C NMR spectroscopy, MALDI TOF mass spec-
trometry, gel permeation chromatography (GPC), and analytical
HPLC are provided in the Supplementary Material (pages S2-3)
[10,29,53,68].
2.4. Dihydrofolate reductase (DHFR) inhibition assay
4.2. Synthesis of MTX-linker 5
Our present studies based on SPR and cell-based binding
experiments provided important pieces of evidence suggesting that
methotrexate molecules attached to the dendrimer carrier could
target FAR for cancer-specific delivery. However, details that define
the mechanisms of drug action after cellular entry are less under-
stood including the release kinetics of MTX inside the cell. Previ-
ously we showed in a cell-free enzyme assay using human DHFR
that this enzyme could be inhibited by free MTX as well as by an
MTX-conjugated PAMAM dendrimer 10 (Ac-G5-FA-MTX) in the
assay condition where MTX molecules are still covalently attached
[63]. In a similar effort to understand the enzyme inhibitory activity
by the current set of dendrimer-MTX conjugates, we chose 7b and
investigated whether it is able to inhibit the human DHFR. In
a standard enzyme assay condition as described elsewhere [63], 7b
potently inhibited the enzyme activity, though less potently than
free MTX (Fig. 7c). This result is in close agreement with our
previous observation from the conjugate 10 [63]. We examined
the stability of 7b against the hydrolytic release of MTX under the
assay condition (PBS, pH 7.4, ambient temperature, incubation
time ¼ 5 min). The stability test showed that MTX was not released
from the conjugate to any meaningful extent (<2% over 48 h). This
indicates that the dendrimer conjugate inhibits DHFR activity even
if MTX molecules are still tethered. The inhibition activity docu-
mented in 7b is supported by numerous structure-activity rela-
tionship studies performed with various types of MTX derivatives.
For example, the activity of human DHFR is potently inhibited by
To a solution of methotrexate hydrate (0.684 mg, 1.45 mmol) in
anhydrous DMF (35 mL) was added cesium carbonate (708 mg,
2.17 mmol), and potassium carbonate (0.3 g, 2.17 mmol). While
stirring this mixture under nitrogen atmosphere, N-(bromoacetyl)-
N0-Boc-1,3-diaminopropane 3 [69] (0.47 g, 1.59 mmol) and sodium
iodide (0.217 g, 1.45 mmol) were added to it. The final mixture was
stirred at room temperature for 48 h under nitrogen atmosphere.
The mixture was concentrated in vacuo, yielding yellow oily
material. It was diluted with 15 mL of 10% MeOH/CH2Cl2, and
purified by flash column chromatography on silica gel (100 g) by
eluting with 10% MeOH/CH2Cl2 to 2% AcOH/15% MeOH/CH2Cl2.
Fractions with an Rf value of 0.27 (2% AcOH in 20% MeOH/CH2Cl2)
were combined and evaporated to afford the desired product 4 as
yellow solid (213 mg, 22%). An HPLC analysis of the purified product
suggested that the regioselectivity of the O-alkylation is 10.2
(g-ester to a d 8.53 (s, 1H),
-ester). 1H NMR (400 MHz, DMSO-d6):
8.45e8.44 (d, NH-Glu, 1H, J ¼ 7.2 Hz), 7.82e7.80 (t, 1H, J ¼ 5.6 Hz),
7.70e7.68 (d, 2H, J ¼ 8.8 Hz), 6.78e6.76 (d, 2H, J ¼ 8.8 Hz),
6.73e6.70 (m, 1H), 4.74 (s, 2H), 4.42 (s, 2H), 4.36e4.35 (m, 1H), 3.17
(s, 3H), 3.02e2.88 (m, 2H), 2.86e2.83 (m, 1H), 1.94e1.85 (m, 1H),
1.48e1.44 (quin, 2H, J ¼ 6.8 Hz), 1.30 (s, 9H) ppm. 13C NMR
(100 MHz, DMSO-d6):
d 174.38, 172.23, 167.36, 166.90, 163.15,
151.58, 149.54, 129.53, 122.0, 120.88, 111.54, 77.94, 62.94, 55.28,
52.69, 36.63, 30.59, 29.90, 28.70, 26.11, 14.36 ppm. MS (ESI): m/z
(relative intensity, %) ¼ 669.3 (100) [M þ H]þ, 569.3 (56) [M-
BocþH]þ. HRMS (ESI) calcd for C30H41N10O8 669.3109, found
669.3123. The N-Boc group in 4 was deprotected by treating with
MTX
g-glutamate esters [64,65] and its poly-g-glutamates [66], the
metabolites that are formed from free MTX in the cell. However we
speculate that this in vitro access is quite different from the in vivo
activity where MTX could be cleaved much faster by cytosolic
esterases and/or by chemical mechanisms after being internalized
to the acidic compartments of the cell [54]. In summary, the
enzyme assay shows potent inhibition of the DHFR by the intact
MTX conjugate as another possible mechanism of drug action in
addition to the MTX release.
TFA. Typically 4 (10 mg, 15 mmol) was mixed with 50% TFA/CH2Cl2
(1 mL) and stirred for 15 min at room temperature. Evaporation of
the solution in vacuo afforded 5 as pale yellow oily residue. It was
used immediately for next step. 1H NMR (400 MHz, CD3OD):
d 8.60
(s, 1H), 7.74e7.72 (d, 2H, J ¼ 7.2 Hz), 6.84e6.81 (d, 2H, J ¼ 7.2 Hz),
4.89 (s, 2H), 4.66e4.62 (d, 1H, J ¼ 15.2 Hz), 4.55e4.51 (d, 1H,
J ¼ 15.2 Hz), 4.53e4.49 (t, 1H, J ¼ 5.6 Hz), 3.34e3.31 (t, 2H,
J ¼ 5.6 Hz), 3.24 (s, 3H), 2.93e2.89 (t, 2H, J ¼ 5.6 Hz), 2.49e2.46 (t,
2H, J ¼ 5.6 Hz), 2.26e2.23 (m, 1H), 2.16e2.12 (m, 1H), 1.88e1.81
3. Conclusion
(quin, 2H, J ¼ 5.6 Hz). 13C NMR (100 MHz, CD3OD):
d 175.20, 171.76,
169.33, 163.51, 156.37, 152.25, 151.75, 148.93, 145.51, 128.99, 121.98,
120.72, 116.86, 115.76, 113.50, 111.34, 62.62, 55.19, 52.94, 36.76,
35.36, 29.85, 29.80, 27.17, 25.49, 15.19 ppm. MS (ESI): m/z (relative
intensity, %) ¼ 569.3 (100) [M þ H]þ, 1137.5 (3) [2M þ H]þ. HRMS
(ESI) calcd for C25H33N10O6 569.2585, found 569.2573.
We investigated
a simplified strategy for cancer-targeted
delivery by using MTX as a dual-acting molecule, serving as
both a chemotherapeutic and a targeting agent. These MTX-
functionalized PAMAM dendrimers effectively kill and also target
cancer cells. Our SPR study proves that the multivalent dendrimer-
MTX conjugate binds FAR on the surface. The cell-based studies
provide evidence that the MTX nanoconjugates are internalized
through FAR because the MTX on the dendrimer cannot get through
the reduced folate carrier because of its nanometer size. In
conclusion, this approach provides novel bifunctional capabilities
for cancer therapy, demonstrates the MTX-mediated cytotoxicity
and targeting to cancer cells, and suggests broad applicability for
other MTX-like pemetrexed and raltitrexed antifolate metabolites
[67] as potential anticancer therapeutics.
4.3. A representative synthesis for G5-MTXn 7b (n ¼ 7.5)
To a suspension of G5e(CO2H)100 6 [53] (50 mg, 1.24
anhydrous DMF (15 mL) was added 4-dimethylaminopyridine
(15.2 mg, 125 mol), N-hydroxysuccinimde (14.4 mg, 125 mol),
and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochlo-
ride (EDC, 23.8 mg, 124 mol). After stirring for 36 h at room
temperature, methotrexate-linker 5 (18.7 mol, 15 eq to G5 den-
drimer) dissolved in 1 mL of DMF and triethylamine (6.3 mg, 50 eq
mmol) in
m
m
m
m

M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
569
to G5 dendrimer) were added to the reaction mixture, and the
mixture continued to be stirred for 24 h at room temperature. At
that time, the mixture was treated with ethanolamine (15.2 mg,
G5-MTXn-FAm 8d (n ¼ 7.5, m ¼ 2.6) was prepared in a manner
similar to 8c otherwise using a higher amount of folic acid-
ethylenediamine amide (1.8 mg, 3.7
mmol). MALDI TOF mass
249
m
mol) and the final mixture was stirred for an additional period
spectrometry: m/z ¼ 48100 gmolꢁ1. Average number of folic acid
covalently attached to the surface of PAMAM G5-glutaric acid is 2.6
(MALDI-based), and 2.0 (1H NMR-based) on a mean basis.
of 6 h. At the conclusion of the reaction, this mixture was
concentrated in vacuo to w3 mL in volume and diluted with 20 mL
of phosphate-buffered saline (PBS, pH 7.2). The solution was loaded
into a membrane dialysis tubing (MWCO 10 kDa), and dialyzed
against PBS (4L ꢀ 2), and deionized water (4L ꢀ 2) over 3 days. The
duration of dialysis performed here was not optimized and could be
reduced to fewer days by replacing each dialysis solution more
frequently until the purity of the dialyzed solution becomes
acceptable by analytical HPLC. The aqueous solution was collected
and lyophilized to afford G5-MTX7.5 as pale yellow solid (37 mg)
and in 98% purity based on analytical HPLC analysis. MALDI
Two other conjugates of this class G5-MTXn-FAm 8a (n ¼ 5,
m ¼ 2) and 8b (n ¼ 5, m ¼ 3.5) were also prepared following the
above procedure for 8c except that different amounts of 5
(15
2.5
m
mol, 12 eq to G5eCO2H) and FA-CONH(CH2)2NH2 (m ¼ 2:
mmol; m ¼ 3.7: 5.0 mol) were used in each of the conjugation
m
reactions [60].
4.5. A representative synthesis for G5-MTXn-FITCp 9b (n ¼ 7.5,
p ¼ 2.4)
TOF mass spectrometry: m/z
¼
47 200 gmolꢁ1
.
GPC:
Mw ¼ 47280 gmolꢁ1, PDI ¼ 1.103. UV/vis (PBS, pH 7.2): 365 (lmax),
G5e(CO2H)100 (50 mg,1.24
reacted with methotrexate-linker 5 (18.7
for 7b. Prior to quenching with ethanolamine, fluorescein-butane-
1,4-diamine [61] (2.4 mg, 5.0 mol) suspended in 1 mL of DMF was
mmol) was activated to NHS ester and
305, 258 nm 1H NMR (400 MHz, DMSO-d6):
d
8.4 (s), 8.05e7.9
mmol) as described above
(broad s), 7.9e7.75 (broad d), 7.7e7.6 (m), 6.75 (broad), 4.75 (s), 4.4
(s), 4.35 (m), 4.2e3.9 (broad), 3.6 (broad s), 3.2e2.8 (m), 2.7e2.0
(m), 1.65 (m), 1.5e1.4 (m) ppm. The resultant nanoconjugate
contains 7.5 methotrexate covalently attached to the surface of
PAMAM G5 dendrimer per mean basis as calculated by dividing the
difference in the molecular weights between two directly compa-
rable conjugates: n (MTX) z½MW7b ꢁ MW6ꢄ=MW5.
m
added to the mixture and stirred for 24 h at ambient temperature.
The mixture was then added with ethanolamine (15.2 mg,
249 mmol) and stirred for additional 6 h. At the conclusion of the
reaction, the mixture was concentrated in vacuo to w3 mL in
volume and diluted with 20 mL of phosphate-buffered saline (PBS,
pH 7.4). The solution was loaded into a membrane dialysis tubing
(MWCO 10 kDa), and dialyzed against PBS (4L ꢀ 2), and deionized
water (4L ꢀ 2) over 3 days. The aqueous solution was collected and
lyophilized to afford G5-MTX7.5-FITC2.4 as pale orange solid (39 mg)
and in 97% purity based on analytical HPLC. MALDI TOF mass
spectrometry: m/z ¼ 48000 gmolꢁ1. UV/Vis (PBS, pH 7.2): 498
Other analogous conjugate G5-MTXn 7a (n ¼ 5) was prepared in
a similar manner otherwise using a lower amount of 5 (15 mmol, 12
eq to 6) while adjusting the amounts of other reagents accordingly.
MALDI TOF mass spectrometry: m/z ¼ 45700 gmolꢁ1. The number
(n) of methotrexate covalently attached to the dendrimer surface is
estimated to be 5, and on a mean basis.
(
lmax), 364, 305, 258 nm 1H NMR (400 MHz, DMSO-d6):
d 8.5 (s),
8.3e8.2 (broad), 8.1e7.9 (broad), 7.9e7.75 (broad d), 7.7e7.6 (m),
7.1e7.0 (m), 6.8e6.65 (broad), 6.6 (s), 6.5e6.4 (m), 4.7 (s), 4.4 (s),
4.3e4.1 (m), 4.1e4.1 (broad), 4.0e3.9 (broad), (broad), 3.75e3.4 (m),
3.2e2.8 (m), 2.7e1.8 (m), 1.65 (m), 1.5e1.4 (m), 1.3 (m) ppm. This
conjugate contains 2.4 fluorescein covalently attached to PAMAM
4.4. A representative synthesis for G5-MTXn-FAm 8c (n ¼ 7.5,
m ¼ 1.2)
G5e(CO2H)100 6 (50 mg, 1.24
ester and reacted with 5 (18.7
Prior to quenching with ethanolamine, FA-CONH(CH2)2NH2 [60]
(0.9 mg, 1.9 mol) suspended in 1 mL of DMF (or dissolved in
m
mol) was activated to its NHS
m
mol) as described above for 7b.
dendrimer on a mean basis: p (FITC) z ½MW9b ꢁ MW7bꢄ=MWFITC
.
The number (p) for attached FITC was also estimated from the
relative ratio between MTX and FITC based on the analysis of 1H
NMR spectrum: p (FITC) z 2.7 on a mean basis.
m
DMSO by brief sonication) was added to the reaction mixture,
and the mixture was stirred for 24 h at ambient temperature, and
followed by treatment with ethanolamine (15.2 mg, 249
G5-MTXn-FIp 9a (n ¼ 5, p ¼ 2) was prepared in a manner similar
m
mol).
to 9b otherwise using a lower amount of 5 (15 mmol, 12 eq to
The final mixture was stirred for additional at room
6
h
G5eCO2H). MALDI TOF mass spectrometry: m/z ¼ 46700 gmolꢁ1
.
temperature, and the mixture was concentrated in vacuo to
w3 mL in volume. After dilution with 20 mL of phosphate-
buffered saline (PBS, pH 7.2), the solution was loaded into
a membrane dialysis tubing (MWCO 10 kDa), and dialyzed
against PBS (4L ꢀ 2), and deionized water (4L ꢀ 2) over 3 days.
The aqueous solution was collected and lyophilized to afford G5-
MTX7.5-FA1.2 as pale yellow solid (35 mg) and in 98% purity
based on analytical HPLC. MALDI TOF mass spectrometry:
The number (p) of fluorescein covalently attached to the surface of
PAMAM G5-glutaric acid is estimated to be 2.3 (MALDI-based), and
2.4 (1H NMR-based) on a mean basis.
4.6. Surface plasmon resonance (SPR) spectroscopy
SPR experiments were performed in BiacoreÒ X (Pharmacia
Biosensor AB, Uppsala, Sweden). Folate binding protein (FBP, bovine
milk) was immobilized on the surface of a CM5 sensor chip through
protein conjugation chemistry to a carboxymethylated dextran-
coated layer on gold following a standard amide coupling protocol
[20]. The immobilizationprocess of FBP on channel 2 resulted in 9500
response unit (RU) equivalent to 9.5 ng/mm2. SPR signals for FBP
binding were obtainedbyinjection of each liganddissolved inHBS-EP
m/z ¼ 47800 gmolꢁ1
. d 8.55
1H NMR (400 MHz, DMSO-d6):
(broad), 8.5 (s), 8.1e7.9 (broad), 7.9e7.75 (broad d), 7.7e7.6 (m),
6.75 (broad), 6.6e6.5 (broad), 4.7 (s), 4.4 (s), 4.35e4.1 (m),
3.9e3.4 (broad), 3.2e2.8 (m), 2.7e2.0 (m), 1.65 (m), 1.5e1.4 (m)
ppm. The nanoconjugate contains 1.2 folic acid covalently
attached to PAMAM G5 dendrimer on a mean basis as calculated
by dividing the molecular weight difference between two
buffer at a flow rate of 30
conjugates 1, 2). After each measurement, the surface of the chip was
regenerated by injection of 10 L of 10 mM glycine-HCl (pH 2.5). SPR
mL/min (FA, MTX), or 50 mL/min (dendrimer
directly comparable conjugates: m (FA) z ½MW8c ꢁ MW7cꢄ=MWFA
.
The number (m) for attached FA could be also estimated from the
1H NMR spectrum where the relative ratio between MTX and FA
was calculated by integration analysis (m z 1.18), and is in good
agreement with the number estimated from the previous mass
data.
m
sensorgrams needed to evaluate binding parameters for each ligand
were obtained from the SPR signals from channel 2 subtracted by
those from channel 1 (
ters, the rate of association (kon), and the rate of dissociation (koff),
D
RU ¼ RU2 ꢁ RU1). Kinetic binding parame-

570
M.-H. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 560e572
were extracted by fitting each binding curve separately using the
Langmuir kinetic model as described [20] in Biacore evaluation
software and were analyzed (dR/dt vs. R) to ensure the absence of the
effect associated with mass transport as discussed elsewhere [70].
Dissociation constants (KD ¼ koff/kon) for each ligand were calculated
by averaging the data obtained from at least three sets of measure-
cytotoxicity experiments, the cells were seeded in 96-well micro-
titer plates (3000 cells/well) in serum-containing medium. Two
days after plating, the cells were treated with different concentra-
tions of MTX or conjugates in tissue culture medium for 4 days.
A
colorimetric XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4-
tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid
hydrate) assay (Roche Molecular Biochemicals, Indianapolis, IN)
was performed following the vendor’s protocol. After incubation
with the XTT labeling mixture, the microtiter plates were read on
an ELISA reader (Synergy HT, BioTek) at 492 nm with the reference
wavelength at 690 nm.
ment per injection concentration which had chi square (
lower than 5.
c
2) values
4.7. Molecular dynamics (MD) simulations
MD simulations on G5 PAMAM dendrimers (2c and 7a) were
carried out using the CHARMM program based on an implicit
solvent model with a 1 fs time step at 300 K [58]. The dendrimer
parameters were obtained from the CHARMM parameters for
generic proteins [59]. The initial structures for the dendrimers
were produced by a recursive script in CHARMM. After steepest
descent and adopted basis NewtoneRaphson minimizations for
10,000 steps, the structures were further annealed at high
temperature and cooled down to room temperature to obtain
lower energy configurations. The systems were equilibrated for
200 ps and then molecular dynamics were carried out for 600 ps.
The total potential energy function (Utotal) for MD simulations is
described as,
4.11. Dihydrofolate reductase (DHFR) assay
MTX and 7b were tested for the inhibitory activity against
human dihydrofolate reductase (Sigma assay kit) according to the
assay protocol as provided [63]. The assay was performed at room
temperature by following the decrease of NADPH and dihydrofolate
concentrations through absorbance measurements at 340 nm at
different concentrations of the inhibitor as indicated in Fig. 5c. The
D
OD (minꢁ1) required for calculating specific enzyme activity
(Units/mg P ¼
m
molmin-1mgꢁ1) in each of the inhibition reactions
was obtained through a linear fitting in the initial phase of reaction
kinetics (0e2.5 min).
Acknowledgment
Utotal ¼ Ubonded þ Unonbonded
ꢀ
ꢃ
hꢁ ꢂ
ꢁ ꢂ i
X X
6
¼ Ubonded
þ
ε
12ꢁ2
þ
qiqj
Dr
1
2
s
s
This work was supported by the National Cancer Institute,
National Institutes of Health under award 1 R01 CA119409 (JRB).
We thank Dr. Pascale Leroueil for the proofreading of this
manuscript.
r
r
i
j
where ε is the minimum energy of the Lennard-Jones potential,
s
the distance yielding a minimum Lennard-Jones potential, q the
partial charge on the atom, D the dielectric constant, r the distance
between i and j and i, j are nonbonded atom pairs. Electrostatic
interactions were modeled by a distance-dependent dielectric
function of the type E(r) ¼ 4r without a long-range nonbonded cut-
off in the simulations [71].
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.11.027.
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