Design considerations for PAMAM dendrimer therapeutics

Article abstract of DOI:10.1016/j.bmcl.2013.03.088

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated

Full text of DOI:10.1016/j.bmcl.2013.03.088

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2872–2875
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design considerations for PAMAM dendrimer therapeutics
a,b,
⇑
a
a
a
a
Sascha N. Goonewardena
, Jeremy D. Kratz , Hong Zong , Ankur M. Desai , Shengzhuang Tang ,
a,b
a
a,
⇑
Sarah Emery , James R. Baker Jr. , Baohua Huang
a
Michigan Nanotechnology Institute for Medicine and Biological Sciences, Ann Arbor, MI 48109, USA
Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine
Received 9 January 2013
Revised 21 March 2013
Accepted 22 March 2013
Available online 1 April 2013
(
PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics
have been difficult to advance because of the complicated syntheses and an incomplete understanding
of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry
to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the tar-
geting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified
MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX
functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Addition-
ally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but
the dendrimer–ester MTX conjugates were much more cytotoxic than the dendrimer–amide MTX conju-
gates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to
design more effective polymer therapeutics.
Keywords:
Cancer
Drug delivery
Dendrimers
Endocytosis
Cleavable linkers
Ó 2013 Elsevier Ltd. All rights reserved.
Because of their controllable properties and carrying capacity,
synthetic polymers have been used for a variety of biomedical
trexate (dendrimer–MTX) conjugates compared to MTX alone.
Our early studies used a sequential syntheses strategy to engineer
the dendrimer–MTX conjugates. Due to inefficiencies in the syn-
thesis, there were inconsistencies in the amount of FA and MTX
1
–3
applications.
Despite these advantages, many polymer thera-
peutics have suffered setbacks because of their complex syntheses
and our limited understanding of the properties responsible for the
1
1
conjugated to the dendrimer scaffold. While these early studies
showed the potential of this approach, the complex synthesis
and a limited understanding of dendrimer properties have slowed
the advancement of these polymer therapeutics. To overcome
some of the synthetic obstacles, we have employed copper-free
strain-promoted alkyne azide cycloaddition (Cu-free click chemis-
4
,5
therapeutic efficacy.
A more complete understanding of the
interactions between synthetic polymers and biological systems
is necessary to more rationally design the next generation of syn-
thetic polymer therapeutics.
Dendrimers are branched, synthetic polymers with controllable
chemical topology that have been used for a broad range of bio-
medical applications including drug delivery, gene transfection,
1
2–15
try) to functionalize the dendrimer scaffold.
The high strain of
the cyclooctyne ring enhances the alkyne reactivity enabling rapid
reaction with azides under mild conditions without the need for
copper catalysts. The efficiency and selectivity of this class of reac-
tions will likely improve the functionalization of dendrimer scaf-
6
and tissue engineering. Because of their low polydispersity and
tunable chemical properties, dendrimers are well suited for mech-
anistic studies on polymer properties and polymer interactions
7
5
with biological systems. Our early studies demonstrated that folic
folds by increasing yields and conjugate consistency.
acid (FA) conjugated to a generation 5 (G5) polyamidoamine (PA-
Herein, we describe the synthesis of dendrimer–MTX conju-
gates using Cu-free click chemistry with variations in the type of
MTX linkage (ester or amide) and targeting ligand (FA or MTX).
Using a human epithelial cancer model, we evaluated the effects
of these dendrimer properties on therapeutic efficacy. Extending
our prior studies to biological systems, we show that both FA
and MTX on the dendrimer scaffold can mediate cell-specific deliv-
ery of therapeutics. Additionally, we demonstrate that the linker-
modified MTX compounds had similar cytotoxicity, but when the
modified-MTX compounds were conjugated to the dendrimer scaf-
fold, there were dramatic differences in therapeutic efficacy.
MAM) dendrimer could target the folate receptor (FR) on cancer
cells to deliver therapeutics in a cell-specific manner.8
–10
Impor-
tantly, these studies demonstrated that cell-specific targeting
greatly enhanced the therapeutic index of the dendrimer–metho-
⇑
Corresponding authors. Tel.: +1 734 615 2139; fax: +1 734 232 4132 (S.N.G.),
tel.: +1 734 615 0618; fax: +1 734 232 4132 (B.H.).
E-mail addresses: sngoonew@med.umich.edu (S.N. Goonewardena), baohua@
med.umich.edu (B. Huang).
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.088

S. N. Goonewardena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2872–2875
2873
Synthesis and characterization of linker modified-MTX
Biological evaluation of linker-modified MTX compounds
compounds and dendrimer-MTX conjugates
To evaluate the therapeutic efficacy of the linker-modified MTX
compounds and the dendrimer–MTX conjugates, we used the An-
nexin V/7-Aminoactinomycin D (An/7AAD) apoptosis assay and
We recently reported the development of a G5 PAMAM dendri-
mer scaffold that can be functionalized using Cu-free click chemis-
try. The linker-modified MTX compounds and the dendrimer–
MTX conjugates were synthesized as shown in Scheme 1. First,
1
2
18
the FR overexpressing human KB cancer cell line. KB cells were
incubated with 100 nM of the linker-modified MTX compounds
1
0
we modified MTX with either an amide-linker (
c-N3-aMTX, 8) or
and free MTX as previously described. At 24 h, KB cells treated
with the linker-modified MTX compounds (8 and 10) and free
MTX all had evidence of apoptosis with both the ester- and
amide-linker MTX compounds treated cells showing approxi-
mately a 100% increase in apoptosis and the MTX treated cells
showing a 200% increase in apoptosis (Fig. 1). At 48 h, the differ-
ence in apoptosis between MTX and the linker-modified MTX com-
pounds was more evident (Fig. 1). Importantly, the modified MTX
compounds retained their cytotoxicity and the ester- and amide-
linker-modified MTX compounds had similar efficacy.
an ester-linker ( -N3-eMTX, 10). To retain its functionality, MTX
c
was modified on the terminal glutamyl functional group preserv-
ing the structural integrity of the pteridine ring and p-aminoben-
1
6
zoic acid (Fig. S1). Because of the structural similarity between
compounds 8, 10, and -azido modified FA, we synthesized the
dendrimer–MTX conjugates by simultaneous reaction with FA
Scheme 1). The dendrimer–MTX conjugates were characterized
c
(
1
by H NMR and had an average of 6 MTX and the FA-dendrimer
conjugates had an average of 3 FA (Fig. S1).
Drug conjugation to polymer scaffolds can be achieved using
stable or cleavable linkages depending on the desired biological ef-
fect. We have used amide and ester bonds to conjugate therapeu-
tics to the dendrimer scaffold. Amide bonds are known to be
extremely stable while ester linkages can be hydrolyzed enzymat-
ically or in acidic or basic environments. On small molecules, ester
linkages hydrolyze quickly and are not as stable compared with
similar amide linkages. Because our approach relies on active tar-
geting and endocytosis, the drug should not be released under nor-
mal physiological conditions that exist outside of the cell. We
monitored the stability of the dendrimer–MTX conjugates in phys-
Biological evaluation of dendrimer-MTX conjugates
After confirming that the linker-modified MTX compounds had
similar activity, we then evaluated the efficacy of the dendrimer–
MTX conjugates. The cytotoxicity of MTX largely depends upon
its ability to inhibit dihydrofolate reductase (DHFR) in the cyto-
1
9
plasm of cells. One challenge for synthetic polymers is to effi-
ciently deliver therapeutic payloads to the cytoplasm of cells.
Cationic polymers can deliver therapeutics to the cytoplasm but
are inherently non-specific and can be cytotoxic themselves.2
To address the issue of cell-specificity, we used FA or MTX as tar-
geting ligands for the dendrimer scaffold. Although this approach
greatly enhances cell specificity because it relies on receptor-med-
iated endocytosis, it results in intracellular compartmentalization
of the therapeutic payload that can lead to changes in therapeutic
efficacy. Based on previous studies, we hypothesized that the den-
drimer–eMTX conjugates would hydrolyze in the endosome allow-
ing MTX to escape into the cytoplasm where it could inhibit the
0,21
1
7
iologic buffers (PBS; pH 7.4) using UPLC as previously described.
Briefly, the four conjugates (2–5) were dissolved in PBS and incu-
bated at 37 °C. At the time points indicated, samples were analyzed
to quantify the amount of free MTX released from the dendrimer–
MTX conjugates. There was no evidence of MTX release from the
dendrimer–amide MTX (dendrimer–aMTX) conjugates (4 and 5)
over 72 h (Fig. S2). The dendrimer-ester MTX (dendrimer–eMTX)
conjugates (2 and 3) also demonstrated excellent stability, releas-
ing approximately 2.3% of the total MTX over 72 h (Fig. S2). This
amount of MTX would not induce cytotoxicity in the biological
experiments. These results demonstrate that the MTX ester and
amide linkages are stable on the dendrimer scaffold in physiologic
buffers and can be used to deliver MTX to cancer cells.
2
2
DHFR. In contrast, due to the stability of the amide linkage, we
hypothesized that the dendrimer–aMTX conjugates would not
hydrolyze in the endosome, trapping MTX and diminishing its abil-
ity to inhibit cytoplasmic DHFR. We evaluated the cytotoxicity of
Scheme 1. Synthesis of linker-modified MTX and dendrimer–MTX conjugates.

2
874
S. N. Goonewardena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2872–2875
the dendrimer scaffold. To evaluate their targeting ability in rela-
tion to the therapeutic activity of the dendrimer conjugates, we
used free FA to block receptor-mediated endocytosis and assessed
the cytotoxicity of the FA and MTX-targeted dendrimer conjugates.
High concentrations of free FA for short periods of time (30 min–
1
h) inhibits FR-mediated uptake of conjugates without affecting
the cytotoxicity of MTX (data not shown). KB cells were incubated
with 100 nM FA for 30 min to saturate the FR and then treated with
1
00 nM of the dendrimer–MTX conjugates for an additional 6 h.
After treatment for 6 h, the cells were washed and fresh media
was added. After 48 h, the cells were assayed for apoptosis using
the An/7AAD assay as described above. Saturation of the FR with
free FA inhibited the cytotoxicity of all of the dendrimer–MTX con-
jugates confirming that the conjugates internalize through the FR
(
Fig. 3). In agreement with our other findings, only cells treated
with the dendrimer–eMTX (2 and 3) showed signs of apoptosis
Fig. 3). At the time points examined, the FA and MTX-targeted
(
Figure 1. Therapeutic activity of linker-modified MTX compounds. KB cells were
treated with 100 nM of MTX, -N3-aMTX (8) and -N3-eMTX (10) and analyzed for
apoptosis at 4, 24, and 48 h. Apoptotic cells were defined as AnV+/7AADÀ and
assessed in relation to a 5% threshold for media control cells. Significance of a 1-
sided paired student t-test recorded (p <0.05), for treatments compared to media
control. Results are representative of three independent experiments.
dendrimer conjugates had similar therapeutic activity that could
be blocked to a similar extent with free FA. These results are in
agreement with recent SPR studies demonstrating that the multi-
valent capacity of the dendrimer scaffold facilitates high avidity
receptor–ligand interactions even when using low-affinity recep-
c
c
2
3
tor–ligand pairs like MTX-FR.
1
00 nM of the dendrimer–MTX conjugates (2–5) at 4, 24, and 48 h.
Therapeutics can be selectively delivered to diseased cells and
tissues by incorporating targeting ligands on the polymer scaffold.
This selectivity can be further enhanced because polymer scaffolds
provide the opportunity for multivalent interactions to enhance
In contrast to the linker-modified MTX compounds, the dendri-
mer–MTX conjugates had striking differences in their cytotoxicity.
At all of the time points evaluated, the dendrimer–eMTX conju-
gates were more cytotoxic compared to the dendrimer–aMTX con-
jugates (Fig. 2). The dendrimer–eMTX 2 demonstrated the greatest
efficacy inducing a ꢀ800% increase in apoptosis relative to the
media control. Similarly, the FA targeted, dendrimer–eMTX conju-
gate (FA dendrimer–eMTX 3) induced a ꢀ400% increase in apopto-
sis relative to the media control. Both of the dendrimer–aMTX
conjugates (4 and 5) had much less activity with only the FA den-
drimer–aMTX 5 conjugate inducing a small amount of apoptosis at
2
4
binding avidity. Previous studies have shown that MTX binds
2
5
the FR with 100-fold lower affinity than FA. Using surface plas-
mon resonance spectroscopy (SPR), we recently showed that deco-
rating the dendrimer surface with MTX greatly enhances the
2
3
conjugate affinity for the FR. Building on these studies, we dem-
onstrate in biological systems that both FA and MTX dendrimer-
conjugates can deliver therapeutic payloads through the FR recep-
tor with similar efficiency. Further studies are under way to vali-
date these findings in vivo. By using MTX as the targeting ligand
and therapeutic, the synthetic scheme for these dendrimer thera-
peutics is greatly simplified which we anticipate will facilitate fur-
ther advancement of these therapeutics.
4
8 h (Fig. 2).
Comparison of FA and MTX as targeting ligands
After evaluating the therapeutic activity of the dendrimer–MTX
conjugates, we compared the targeting ability of MTX and FA on
Figure 3. Competition experiments to compare folate receptor targeting of
dendrimer FA and MTX ligands. KB cells were incubated with or without 100 nM
free FA for 30 min and then treated with 100 nM of the dendrimer–MTX conjugates
(2–5) for 6 h. After 6 h, cells were washed and media was replaced. KB cells were
assayed at 48 h for AnV+/7AADÀ as a marker of apoptosis as above. Apoptotic cells
were defined as AnV+/7AADÀ and assessed in relation to a 5% threshold for media
Figure 2. Therapeutic activity of dendrimer–MTX conjugates. KB cells were treated
with 100 nM of the dendrimer–MTX conjugates (2–5) and analyzed for apoptosis at
4
, 24, and 48 h. Apoptotic cells were defined as AnV+/7AADÀ and assessed in
⁄
relation to a 5% threshold for media control cells. Significance of a 1-sided paired
student t-test recorded (p <0.05), for treatment groups compared to media control.
Results are representative of three independent experiments.
control cells. Significance of a 1-sided paired student t-test recorded ( p <0.05 and
⁄
⁄
p <0.01), for treatment groups versus media control. Results are representative of
three independent experiments.

S. N. Goonewardena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2872–2875
2875
We and others have shown that therapeutics can be incorpo-
rated on the dendrimer scaffold through either complexation or
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This project has been funded with funds from the National Can-
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S.N.G. was supported by an Inaugural Grant from the University of
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Supplementary data
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Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2
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