Advanced Drug Delivery Reviews 57 (2005) 2203 – 2214

www.elsevier.com/locate/addr

Targeted drug delivery with dendrimers: Comparison of the release

kinetics of covalently conjugated drug and non-covalent drug
inclusion complexB
Anil K. Patri, Jolanta F. Kukowska-Latallo, James R. Baker Jr. *
Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan Health System, Ann Arbor, MI 48109-0648, USA
Received 3 March 2005; accepted 13 September 2005
Available online 14 November 2005

Abstract

Dendrimers have unique characteristics including monodispersity and modifiable surface functionality, along with highly
defined size and structure. This makes these polymers attractive candidates as carriers in drug delivery applications. Drug delivery
can be achieved by coupling a drug to polymer through one of two approaches. Hydrophobic drugs can be complexed within the
hydrophobic dendrimer interior to make them water-soluble or drugs can be covalently coupled onto the surface of the dendrimer.
Using both methods we compared the efficacy of generation 5 PAMAM dendrimers in the targeted drug delivery of methotrexate
coupled to the polymer. The amine-terminated dendrimers bind to negatively charged membranes of cells in a non-specific manner
and can cause toxicity in vitro and in vivo. To reduce toxicity and to increase aqueous solubility, modifications were made to the
surface hydroxyl groups of the dendrimers. For targeted drug delivery, the dendrimer was modified to have a neutral terminal
functionality for use with surface-conjugated folic acid as the targeting agent. The complexation of methotrexate within a
dendrimer changes the water insoluble drug into a stable and readily water-soluble compound. When this dendrimer complexed
drug, however, was placed in a solution of phosphate buffered saline, the methotrexate was immediately released and displayed
diffusion characteristics identical to free methotrexate. Covalently coupled methotrexate dendrimer conjugates were stable under
identical conditions in water and buffered saline. Cytotoxicity tests showed that methotrexate as the dendrimer inclusion complex
had an activity identical to the free drug in vitro. In contrast, folic acid targeted dendrimer with covalently conjugated methotrexate
specifically killed receptor-expressing cells by intracellular delivery of the drug through receptor-mediated endocytosis. This
study demonstrates that while drug as a dendrimer inclusion complex is readily released and active in vitro, covalently conjugated
drug to dendrimer is better suited for specifically targeted drug delivery.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Dendrimers; Drug delivery; Inclusion complex; Methotrexate; Folic acid; Folate receptor

B
This review is part of the Advanced Drug Delivery Reviews theme issue on bDendrimers: a Versatile Targeting PlatformQ, Vol. 57/15, 2005.
* Corresponding author. Department of Internal Medicine Chief, Division of Allergy and Clinical Immunology, Nanotechnology Institute for
Medicine and Biological Sciences, 9220 MSRB III Ann Arbor, MI 48109-0648, USA. Tel.: +1 734 647 2777; fax: +1 734 936 2990.
E-mail address: jbakerjr@umich.edu (J.R. Baker).
URL: http://www.nano.med.umich.edu (J.R. Baker).

0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2005.09.014
2204 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2204
2. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2205
2.1. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2205
2.2. Cell culture and cytotoxicity measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2206
2.3. Synthesis of dendrimer conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2206
2.3.1. G5–FA–MTX conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2206
2.3.2. G5–MTX conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2206
2.3.3. Inclusion complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2207
2.4. Gel filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2207
3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2207
3.1. Methotrexate release studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2209
3.2. Targeted cytotoxicity of MTX–dendrimer inclusion complexes and conjugates . . . . . . . . . . . . . 2209
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212

1. Introduction ity. This has led to investigations of novel carrier

Targeted drug delivery directs a therapeutic agent
in a site-specific manner to a particular cell type or
tissue. It could be beneficial in cancer therapy where
cytotoxic drugs can kill tumor cells while sparing
healthy tissue overcoming the toxic side effects
associated with chemotherapy. In cancer therapy
several methods have been proposed to target tumors.
One employs the enhanced permeability and retention
(EPR) phenomenon in tumors [1] where microvascu-
lature allows permeation of molecules and the absence
of lymphatic drainage leads to retention and accumu-
lation of the delivered compounds in a tumor. This has
the potential to concentrate drugs to some extent in
tumors when using polymeric drug carriers of a size
that pervade tumor vasculature [2,3]. In EPR target-
ing, the therapeutic agent is subsequently released
locally and taken up by tumor cells to achieve its
effect. In contrast, tumor receptor or antigen targeting
specifically aims the therapeutic agent at tumor cells.
Targeted tumor cells express or over-express unique
receptors or antigens. Once identified, they can be
targeted using antibodies, small molecules or peptides
that can recognize and bind to the sites. In this
approach, a therapeutic or a bioactive agent can be
coupled directly to a targeting moiety for selective
delivery. There are limitations, however, posed by
these conjugates, such as reduced specificity, de-
creased solubility, and limited drug carrying capabil-
systems [4] that overcome the problems, have longer
circulation times and offer the potential to carry larger
amounts of drug. The aim of these applications is that
the macromolecular carrier remains soluble and
complexed to both the therapeutic and targeting
agents until it reaches the targeting site. Any
degradation of the carrier or premature release of free
drug before reaching the desired location defeats the
purpose of the carrier and reduces targeting efficacy,
leading to increased side effects. For this reason, a
stable system is required for any macromolecular
carrier system for targeted drug delivery.
Dendritic polymers, or dendrimers, have been
proposed as one type of carrier for use in targeted
drug delivery [5,6]. The unique characteristics of
dendrimers such as uniform and controlled size,
monodispersity, and modifiable surface group func-
tionality make these molecules appealing for biomed-
ical applications [5–7]. The ability to functionalize
their terminal groups with various targeting, thera-
peutic, and imaging agents in a specific and control-
lable manner leads to the potential to use them as
carriers for targeted drug delivery. The utility of the
internal void volume of dendrimers to encapsulate
hydrophobic guest molecules and drugs has been
demonstrated by several research groups [8–11]. The
resulting problems with aqueous solubility, biocom-
patibility, and availability, however, have limited drug
delivery investigations to very few classes of den-
 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214
drimers. The PAMAM dendrimers, which fulfill most
requirements for use in in vivo applications are being
considered extensively for medical applications
[12,13]. PAMAM dendrimers are being investigated
as carriers in gene transfection [14], MRI contrast
agents[15–17], boron–neutron capture therapy
[18,19], and drug delivery applications [7,20,21].
The internal tertiary amines of PAMAM dendrimers
are available for acid–base interactions and hydrogen
bonding as well as for other non-covalent interactions
with encapsulated guest molecules, thus making the
polymers effective agents for solubilizing hydropho-
bic drugs [22,23].
Research efforts on drug encapsulation in den-
drimers led to the synthesis of polyethylene glycol
(PEG) functionalized dendrimers to impart aqueous
solubility, biocompatibility, and increased drug
loading with greater size of the dendrimer [24,25].
Ibuprofen encapsulated, G3 and G4 amine function-
alized PAMAM dendrimers were investigated for
their uptake into cells [26]. In all these efforts,
however, the encapsulated drug molecules were
released readily in isotonic solutions, rendering
them ineffective as carriers for targeted drug
delivery. On the other hand, efforts towards
covalent attachment of drugs to the surface of
polyaryl ether dendrimers resulted in the conju-
gates’ becoming insoluble at physiological pH [27].
Addition of the PEG (polyethylene glycol) function
to polyaryl ether dendrimers limited the solubility
of drug–dendrimer conjugates at physiological con-
ditions because of the hydrophobic aromatic den-
dritic architecture coupled with hydrophobic drugs
on the surface [28].
We have investigated targeted drug delivery
using a PAMAM dendrimer platform. Modification
of the positively charged terminal amine function-
ality to neutral, acetylated or hydroxylated surfaces
decreased non-specific uptake of these dendrimers,
improved aqueous solubility of various conjugates,
and reduced aggregation [29,30]. Highly reproduc-
ible results for targeting with folic acid (FA), both
in vitro and in vivo, encouraged further investiga-
tions into imaging and drug delivery [31]. In the
current study, we synthesized highly soluble tar-
geted dendrimer conjugates and compared the
efficacy of covalently bound methotrexate (MTX)
with an MTX inclusion complex utilizing genera-
2205
tion 5 PAMAM dendrimer. The terminology
binclusion complexQ was used to depict dendrimer
containing a free drug bound by non-covalent
interaction and bconjugateQ was used to represent
a drug covalently conjugated to dendrimer. The
drug release characteristics from unmodified
PAMAM dendrimer–inclusion complexes and cova-
lently conjugated drugs are evaluated in vitro for
the first time under the conditions replicating those
necessary for drug delivery.
2. Experimental
2.1. Materials and methods
Folic acid, methotrexate, EDC, glycidol, hydrox-
yl terminated PAMAM dendrimer, methanol and
dimethyl sulfoxide were purchased from Aldrich
(St. Louis, MO). An Isco Low Pressure Liquid
Chromatography (LPLC) system with UA-6 UV-Vis
detector and type 11 (254 nm) or type 12 (214 nm)
optical units in conjunction with a Sephadex G-25
column was used for purification and detection of
eluents by gel filtration. UV measurements were
carried out on a Perkin Elmer Lambda 20
spectrometer and NMR measurements on a Bruker
500 MHz spectrometer. Generation 5 PAMAM
dendrimers were synthesized as described below
and characterized by 1H and 13C NMR, GPC, and
MALDI-TOF Mass Spectrometry.
Unless specified, purification of all the dendrimer
conjugates was carried out by dialysis in a 10 K
MWCO regenerated cellulose bag (Slide-A-Lyzer
dialysis cassette; Pierce, Rockford, IL), with the initial
buffer exchanges performed with phosphate buffered
saline (PBS pH 7.4) and then several times with
deionized water. The release of methotrexate from
either an inclusion complex or a covalent conjugate
was monitored by UV spectroscopy. The inclusion
complex or conjugate was introduced into a Micro
DispoDialyzerk (8 kD MWCO; Spectrum Laborato-
ries Inc., Rancho Dominguez, CA) and dialyzed in
PBS or water directly into a cuvette while stirring. The
UV spectra of permeates were taken at 15-min
intervals, and the absorbance was compared from a
concentration curve of methotrexate to determine the
amount of released methotrexate.
2206 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214

2.2. Cell culture and cytotoxicity measurement O

a N C FA
NH2 H
KB is a human cell line that over-expresses folate 12 8
NH2
receptor (FAR) when grown in folate-deficient medium G5-NH2 G5-FA-NH2
[32]. Cells were purchased from the American Type
Culture Collection (ATCC, Manassas, VA) and main- b c O
tained at 37 8C with 5% CO2 in folate-deficient RPMI C FA
NH
1640 supplemented with penicillin (100 units/mL), N OH
streptomycin (100 Ag/mL), and 10% heat-inactivated HO
2 N OH
FBS. For toxicity studies cells were plated in 96-well G5-OH HO
G5-FA 2
plates at 5000 cells/well in 100 AL of medium. After 24
h incubation of cells, conjugates were applied in 5 AL d
of medium at a final concentration range from 1 nM to
d O
C FA
50 AM of MTX. The concentration of compounds was NH
N OH
adjusted based on the number of MTX molecules HO
RN 2
present in the inclusion complex or conjugate. The N OH
RN HO
control dendrimers without MTX were used at the HO
2

molar concentrations of dendrimer present in the HO

inclusion complex and/or conjugate. Cells were pre-
incubated with the conjugates for 1.5 h, then the
medium was removed and the cells were washed with
additional volumes of culture medium. After additional
incubation for 72 h cell viability was determined using
a colorimetric XTT based assay (Roche Diagnostics,
Indianapolis, IN). The assay measures the conversion
of XTT (sodium 3V-[1-(phenylamino-carbonyl)-3,4-
tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic
acid hydrate] labeling reagent) into water soluble
formazan salt by mitochondrial dehydrogenase activity
of viable cells [33]. The absorbance of the samples was
measured at 492 nm using a SPECTRA MAX 340
microtiter plate reader (Molecular Devices Corp.,
Sunnyvale, CA).
2.3. Synthesis of dendrimer conjugates
2.3.1. G5–FA–MTX conjugate
Generation 5 amine terminated PAMAM dendrimer
G5–NH2 (Scheme 1) was used for the current studies.
The synthesis of folic acid conjugated dendrimer G5–
FA–NH2 was carried out using a previously published
procedure [29]. The unreacted folic acid and urea
byproducts were removed by ultrafiltration using a
Pellicon device. To a solution of G5–FA–NH2 (250 mg;
8.9 Amol) in a 1 : 1 mixture of DMSO / pH 8.5 PBS (8
mL), excess glycidol (158 mg; 2.1 mmol) was added
and stirred for 3 h. An additional aliquot (158 mg; 2.1
mmol) of glycidol was added and after 3 h the resultant
O
O
MTX
O MTX
O
G5-MTX G5-FA-MTX
Scheme 1. Synthesis of dendrimer conjugates. a) Folic acid, EDC,
DMSO; b) Glycidol, DMSO; c) Glycidol, DMSO/PBS; d)
Methotrexate, EDC, DMSO. Conjugates 3 and 4 were used to
make inclusion complexes. Conjugates 5 and 6 are covalent
conjugates.
mixture was dialyzed and lyophilized to yield 280 mg
of the folate–hydroxyl–terminated dendrimer G5–FA.
Methotrexate (7 mg; 15.3 Amol) was activated with
EDC (4 mg; 18.4 Amol) for 30 min in DMSO and added
drop-wise under argon to a solution of G5–FA (66 mg;
1.5 Amol) in 5 mL of anhydrous DMSO and stirred for
8 h at room temperature. The resultant mixture was
purified by dialysis and lyophilized, yielding 60 mg of
purified conjugate G5–FA–MTX.
2.3.2. G5–MTX conjugate
To a solution of generation 5 PAMAM dendrimer
G5–NH2 (250 mg; 10 Amol) in anhydrous DMSO (5
mL) excess glycidol (300 mg; 4 mmol) was added and
stirred at room temperature under nitrogen. The
reaction mixture was then dialyzed. The purified
conjugate was lyophilized and yielded 260 mg of
G5–OH conjugate. Methotrexate (8.1 mg; 17.82 Amol)
was activated with EDC (4.1 mg; 21 Amol) in
anhydrous DMSO (0.5 mL) for 30 min, added drop-
 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214
wise to a solution of G5–OH (50 mg; 1.2 Amol) in
DMSO (3 mL) and stirred for 8 h under nitrogen. The
mixture was dialyzed and lyophilized, yielding 53 mg
of G5–MTX conjugate.
2.3.3. Inclusion complex
Dendrimer inclusion complexes were prepared by
adding 0.6 Amol of methotrexate to 66 nmol of
dendrimer solution in deionized water (0.5 mL),
vortexing followed by stirring for 2 h and then
lyophilizing the resultant inclusion complex. The
complex was then re-dissolved in 0.2 mL of deionized
water and an aliquot of 50 AL was dialyzed (8 kD
MWCO) at room temperature as described earlier and
the cumulative release of methotrexate was moni-
tored. Alternately, the release studies were carried out
in a dialysis cassette (10 K MWCO) as described
above. This method, requiring a larger amount of
material, had a faster release rate.
2.4. Gel filtration
Gel filtration of the dendrimer inclusion complexes
and covalently coupled conjugates were conducted on
a G-25 sephacryl column pre-equilibrated and eluted
with phosphate buffered saline. Samples were applied
to the column, and eluting fractions were detected at
210 nm and isolated using a fraction collector.
3. Results and discussion
The use of folic acid-conjugated PAMAM den-
drimer for targeting and imaging applications was
reported previously by several groups including ours
[15,30,34]. The well-documented conjugation of folic
acid to dendrimer was chosen to test different delivery
approaches through targeting. When limited numbers
of folic acid molecules (5 per dendrimer) are
conjugated to G5 PAMAM dendrimer, the conjugate
retains solubility in water [30]. The use of phosphate
buffered saline solution (PBS) during the initial phase
of ultrafiltration removes complexed small molecules
from the inclusion complex, but does not disrupt the
covalent drug linkage. Subsequent washings with
deionized water remove the buffer salts. This proce-
dure of purification assures a high purity of covalent
dendrimer conjugate. The G5–FA–NH2 conjugate was
2207
characterized using NMR and UV spectroscopy and
was determined to have an average of 5 folic acid
moieties per dendrimer. The surface of the dendrimers
was modified to achieve neutral (acetylated or
hydroxylated substitution) or negatively charged
(carboxylic acid substitution) terminal group function-
alities because the amine-terminated dendrimers bind
non-specifically to cells. The binding characteristics
of all of the modified dendrimers, with or without
conjugated folic acid, were compared using a folic
acid receptor (FAR) expressing cell line [30]. The
acetylated and hydroxylated dendrimers were both
found to have low non-specific binding and high
aqueous solubility. Although the conjugation of
hydrophobic groups to dendrimers decreases their
aqueous solubility, the hydroxyl-functionalized den-
drimers impart aqueous solubility even after partial
conjugation with hydrophobic groups. The hydroxyl
groups on the surface facilitate ester conjugation of
the carboxylic acid containing drugs as well. These
ester conjugates are acid labile and are stable in
neutral buffers. We have chosen to test the efficacy as
a difference in cytotoxicity measured in vitro of the
drug delivered in the form of ester-conjugated MTX
or as an inclusion complex containing MTX.
The folate–dendrimer conjugate G5–FA–NH2 was
reacted with excess glycidol to functionalize the
terminal primary amines to a hydroxyl-terminated
G5–FA dendrimer. This conjugate is highly soluble in
aqueous buffers. The MTX was then conjugated to G5–
FA by EDC coupling in anhydrous DMSO to form an
ester-linked MTX–dendrimer–FA conjugate (G5–FA–
MTX). This conjugate was purified by dialysis. The 1H
NMR in D2O had broad overlapping signals in the
aliphatic region between d 4 to 2 ppm corresponding to
the dendrimer C H 2 protons and aromatic signals at d
6.7, 7.6, and 8.5 ppm. The UV spectrum had over-
lapping absorptions due to the presence of both FA and
MTX (Fig. 2b) covalently bound to dendrimer, which
are different from conjugates containing only FA or
MTX. As a control without FA, generation 5 PAMAM
dendrimer was surface-functionalized with glycidol to
give the hydroxyl-terminated dendrimer G5–OH. The
MTX was conjugated to this dendrimer using EDC
coupling as described earlier and purified by dialysis.
The 1H NMR of this G5–MTX conjugate showed
overlapping broad signals in the aliphatic region
between d 4–2 ppm for the dendrimers methylene
2208 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214

protons and three signals in the aromatic region
between d 6.6 to 8.5 ppm. The UV spectrum of this
conjugate has a characteristic absorption similar to that
of free MTX in addition to an intense absorption at 210
nm for the dendrimer, confirming the conjugation.
Dendrimer conjugates were tested for purity by gel
filtration using LPLC. Initially the MTX inclusion
complex with G5–FA conjugate was filtered through a
Sephadex G-25 column using LPLC. Two peaks were
detected (Fig. 1a) and analyzed by UV spectroscopy
(Fig. 1b). As expected, the first fraction with
absorbance was analyzed to be the dendrimer–FA
conjugate (G5–FA) with characteristic absorption for
FA at ODmax 286, while the second fraction appeared
to be the free MTX. This indicates that charge-based
drug inclusion complexes can be disrupted by
phosphate-buffered saline solution and that the den-
drimer–drug conjugates can be purified by gel-
filtration. The dendrimer conjugate with covalently
coupled FA and MTX (G5–FA–MTX) was stable

b
1

0.9

0.8

0.7
Fraction 1
Absorbance

0.6 Fraction 2
0.5

0.4

0.3

0.2

0.1

0
200 250 300 350 400 450 500
Wavelength (nm)

Fig. 1. (a) LPLC chromatogram of methotrexate inclusion complex with G5–FA upon gel filtration. First isolated fraction was G5–FA conjugate
and the second, later eluted fraction was free methotrexate (Fig. 1b). (b) UV spectra of two fractions isolated upon gel filtration of a dendrimer–
methotrexate inclusion complex. The UV spectrum of fraction 1 is identical to the G5–FA conjugate and fraction 2 is identical to that of free
methotrexate.
 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214
under the gel-filtration conditions and did not yield
any free methotrexate. The only fraction detected (Fig.
2a) was analyzed by UV spectroscopy (Fig. 2b) and
contained the dendrimer with both FA and MTX. The
non-targeted dendrimer–MTX conjugate (G5–MTX)
without folate was also found to be stable and did not
contain detectable free MTX (data not shown).
3.1. Methotrexate release studies
The dendrimer–drug inclusion complex is soluble
and stable in water with minimal release of free MTX
(less than 5%) when dialyzed against water. The
hydroxyl-terminated G5 dendrimer G5–OH, however,
releases MTX from its inclusion complex when
a enough to retain MTX in an inclusion complex when
b into cells after ligand-binding [36,37]. We have
1.2
1 and internalization in vitro of PAMAM dendrimers
Fraction 1
Absorbance
0.8
0.6
0.4
0.2
0 antineoplastic drug methotrexate (MTX) is a folic
200 300 400 500
Wavelength (nm)
Fig. 2. (a) LPLC chromatogram of a G5–FA–MTX conjugate. A
single fraction was isolated and no detectable free methotrexate was
observed. (b) UV spectrum of the single fraction isolated upon gel
filtration of a G5–FA–MTX conjugate. The spectrum shows
absorption for both folic acid and methotrexate.
2209
dialyzed in PBS (Fig. 3). More than 70% of the
included MTX was detected in PBS within 2.5 h. The
release rate of free MTX from the dialyzer in PBS did
not differ from MTX released from the dendrimer
inclusion complex under the same conditions. The
release rate of MTX from the inclusion complex with
G5–FA showed a similar pattern, with 50% of the
drug being released from the dialyzer in PBS in
2.5 h whereas there was no release of MTX from this
compound when dialyzed in water. This trend of
MTX release from the inclusion complexes with
dendrimers in PBS is consistent and reproducible
with either hydroxyl, acetylated, or amine surface
dendrimers, although the rate of release is slightly
different, depending on the method and the surface
composition. This indicates that the interactions
between the dendrimer and MTX are not strong
the inter-molecular forces are neutralized in a
buffered salt solution. In contrast MTX covalently
conjugated to dendrimer is neither released in water
nor in phosphate-buffered saline.
3.2. Targeted cytotoxicity of MTX–dendrimer inclu-
sion complexes and conjugates
Targeting through the high-affinity receptor (FR)
for the vitamin folic acid (FA), also known as the
folate binding protein, has been achieved by delivery
of carriers with conjugated folic acid [35]. The FR is
over-expressed in many cancers and is internalized
previously demonstrated the highly selective binding
covalently conjugated with FA in KB tumor cells that
over-express FR [30]. The KB tumor human cell line
over-expresses the FR when maintained in a folate-
deficient medium [38]. This FR expression can be
down regulated by culturing KB cells in a thousand-
fold excess of FA or can be transiently blocked by the
addition of free FA into the medium [30]. The
acid antagonist with several-hundred-fold lower af-
finity for FR than folic acid [39–41]. The cytotoxicity
of inclusion complexes obtained with KB cells over-
expressing FR (FAR+) is similar to the cytotoxicity of
free MTX in a concentration range of drug from 0.05
to 0.1 AM (Fig. 4). The MTX is released from
2210 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214

Release Profile of MTX from various Dendrimer Conjugates and Inclusion

Complexes in both PBS and Water
100
MTX in PBS
90

80 MTX inclusion with

G5-OH in PBS
70
MTX inclusion with
Percent Released

60 G5-FA in PBS
G5-FA-MTX conjugate
50
in PBS
40 G5-MTX conjugate in
PBS
30
MTX inclusion with
20 G5-OH in water
10 MTX inclusion with
G5-FA in water
0
0 0.5 1 1.5 2 2.5 3
Time (hrs)

Fig. 3. Comparative release profile of methotrexate (MTX) from inclusion complexes and conjugates in water and phosphate-buffered saline
(PBS). Methotrexate in inclusion complexes in PBS was released to the same degree as free MTX. The MTX inclusion complexes in water and
covalent conjugates in PBS did not release free methotrexate.

KB (FAR+) 1.5h preincubation

120
MTX

100 G5-OH control

Cell Viability (% control)

G5-FA control
80
G5-MTX
conjugate
60
G5-FA-MTX
conjugate
40 MTX Inclusion
with G5-OH
MTX Inclusion
20 with G5-FA
Control
0
0.001 0.01 0.1 1 10 100
Concentration (µM)

Fig. 4. Cytotoxicity of covalent conjugates with MTX and dendrimer inclusion complexes with MTX tested on KB cells over-expressing FA
receptor (FAR+). Cells were pre-incubated for 1.5 h. with methotrexate containing dendrimers, and dendrimers without drug, washed with medium
and further incubated for 72 h. While the dendrimer control conjugates G5–OH and G5–FA without methotrexate are not cytotoxic, the
methotrexate inclusion complexes with G5–OH and G5–FA were as equally toxic as free methotrexate in a concentration range of MTX from 0.05
to 0.1 AM. The G5–MTX conjugate without folic acid is cytotoxic at higher (about 1.0 AM) concentration of MTX, compared to the targeted G5–
FA–MTX conjugate at 0.3 AM of MTX.
 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214 2211

KB (FAR+) 1.5h preincubation blocked with free FA

120
MTX

100 G5-OH
control

G5-FA control
Cell viability (% control)

80
G5-MTX
conjugate
60
G5-FA-MTX
conjugate

40 MTX inclusion
with G5-OH
MTX inclusion
20 with G5-FA

Control

0
0.001 0.01 0.1 1 10 100
Concentration (µM)

Fig. 5. Specificity of delivering FA-targeted MTX conjugates and inclusion complexes. Cytotoxicity was tested using KB (FAR+) cells after
initial blocking of cell culture with free folic acid (9 AM) followed by the pre-incubation with compounds for 1.5 h, wash and further incubation
for 72 h. The free methotrexate and inclusion complexes were toxic only at a higher (10 fold) concentrations of MTX (1 AM) compared to non-
blocked KB (FAR+) cells (Fig. 4). While the G5–FA–MTX conjugate showed toxicity at 10.0 AM of MTX, the control non-targeted G5–MTX
conjugate was not toxic at the same concentration of MTX.

KB (FAR-) 1.5 h preincubation

120
MTX

100 G5-OH control

Cell Viability (% control)

80 G5-FA control

G5-MTX
60
conjugate
G5-FA-MTX
40 conjugate
MTX inclusion
20 with G5-OH
Control

0
0.001 0.01 0.1 1 10 100
Concentration (µM)

Fig. 6. Specificity of delivering MTX via FA receptor using KB cells with low expression of the FA receptor (FAR ). Compared with Fig. 5, a
10-fold higher concentration of drug was required to achieve similar cytotoxicity. Compared to the FAR (+) cells, methotrexate was toxic at
higher concentrations (N1 AM) and similar to the MTX in the inclusion complex. The G5–FA–MTX and G5–MTX conjugates had toxicity at
N10 AM concentration of MTX.
2212 A.K. Patri et al. / Advanced Drug Delivery Reviews 57 (2005) 2203–2214
inclusion complexes within 1.5 h of pre-incubation
with cells and is as effective as free MTX. The effective
cytotoxic concentration of the conjugated MTX was
from 0.3 to 1.0 AM (Fig. 4). Similar patterns of
cytotoxicity but at 10- to 20-fold higher concentrations
of MTX were observed for inclusion complexes (1.0
AM) and the conjugates (10.0 AM) when the initial 1.5
h uptake was blocked with an excess of free folic acid,
indicating receptor-mediated uptake (Fig. 5). The
specific uptake of covalently conjugated methotrexate
through receptor-mediated internalization has to be
followed by the hydrolysis of free drug to effect
cytotoxicity. The ester-linked MTX, FA-targeted con-
jugate G5–FA–MTX is required at about 10-fold higher
concentration to have the same effect as the inclusion
complex as the result of the slower release of the
conjugated MTX after receptor-mediated internaliza-
tion of the conjugate (Figs. 4 and 5). In contrast, much
smaller inhibition from folic acid was observed with
the drug inclusion complex and free MTX (Fig. 5).
These observations were further confirmed using KB
cells with down-regulated FR (FAR ). The toxicity
pattern remained similar in inclusion complexes and
conjugates and at the very high concentrations of MTX
(5.0–10.0 AM) the differences could not be clearly
discerned (Fig. 6).
4. Conclusion
We have synthesized generation 5 PAMAM
dendrimer conjugates with folic acid and modified
surface to give respectively a hydrophilic, neutral,
hydroxyl surface. Methotrexate inclusion complexes
were formed in water and their cytotoxicity was
compared with MTX covalently conjugated by an
ester linkage that mediates release of the active
drug through hydrolysis. Even though free metho-
trexate is retained within the drug complex when
dialyzed against deionized water, it is readily
released in phosphate-buffered saline (PBS) and,
in the cell culture, is available in a manner similar
to the free drug. The activity of MTX is
proportional to the number of MTX molecules in
the inclusion complex. All the covalently coupled
drug–dendrimer conjugates were stable in both
water and PBS buffer solution. This suggests that,
while complexing a drug with dendrimer as an
inclusion complex improves its solubility in water,
a cleavable, covalently linked dendrimer conjugate
is better suited for targeted drug delivery as it does
not release the drug prematurely in biological
conditions. Although in the in vitro cell culture
assays used here, the cytotoxicity of the inclusion
complex is identical to the free methotrexate on
FAR (+), FAR (+) blocked with free folic acid, and
FAR ( ) cells, the viability of the cells depends on
the presence of the receptor. The specificity of the
internalization of the covalent conjugate is clearly
discernable on KB cells over-expressing FR and is
further supported by the blocking of toxicity with
the free folic acid or by the lack of cytotoxic effect
in FAR ( ) cells.
Acknowledgements
We thank Sonya Krasilshchikova, David Leavitt,
Adam Was and Andrea East for their technical
assistance. This project has been funded in whole
or in part with Federal Funds from the U.S. Army
Medical Research and Material Command, Depart-
ment of Defense, under grant DAMD17-02-1-0096
and National Cancer Institute, National Institutes of
Health, under Contract #NOI-CO-97111.
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