Cytotoxicity, hemolysis, and acute in vivo toxicity of dendrimers based on melamine, candidate vehicles for drug delivery

Article abstract of DOI:10.1021/ja048548j

A small library of dendrimers was prepared from a common precursor that is available in 5 g scale in five linear steps at 56% overall yield. The precursor is a generation three dendrimer that displays 48 peripheral sites by incorporating AB₄ surface groups. Manipulation of these sites provided six dendrimers that vary in the chemistry of the surface group (amine, guanidine, carboxylate, sulfonate, phosphonate, and PEGylated) that were evaluated for hemolytic potential and cytotoxicity. Cationic dendrimers were found to be more cytotoxic and hemolytic than anionic or PEGylated dendrimers. The PEGylated dendrimer was evaluated for acute toxicity in vivo. No toxicity-neither mortality nor abnormal blood chemistry based on blood urea nitrogen levels or alanine transaminase activity-was observed in doses up to 2.56 g/kg ip and 1.28 g/kg iv.

Full text of DOI:10.1021/ja048548j

Published on Web 07/22/2004
Cytotoxicity, Hemolysis, and Acute in Vivo Toxicity of
Dendrimers Based on Melamine, Candidate Vehicles for Drug
Delivery
Hui-Ting Chen,† Michael F. Neerman,† Alan R. Parrish,*,‡ and Eric E. Simanek*,†
Contribution from the Department of Chemistry, Texas A&M UniVersity, and Department of
Medical Pharmacology and Toxicology, Texas A&M UniVersity Health Science Center,
College Station, Texas 77843-3255
Received March 12, 2004; E-mail: simanek@tamu.edu
Abstract: A small library of dendrimers was prepared from a common precursor that is available in 5 g
scale in five linear steps at 56% overall yield. The precursor is a generation three dendrimer that displays
48 peripheral sites by incorporating AB4 surface groups. Manipulation of these sites provided six dendrimers
that vary in the chemistry of the surface group (amine, guanidine, carboxylate, sulfonate, phosphonate,
and PEGylated) that were evaluated for hemolytic potential and cytotoxicity. Cationic dendrimers were
found to be more cytotoxic and hemolytic than anionic or PEGylated dendrimers. The PEGylated dendrimer
was evaluated for acute toxicity in vivo. No toxicitysneither mortality nor abnormal blood chemistry based
on blood urea nitrogen levels or alanine transaminase activityswas observed in doses up to 2.56 g/kg ip
and 1.28 g/kg iv.
suggesting that higher dosing might be attainable.6a In vitro and
in vivo studies have shown that cationic dendrimers based on
Introduction
As the products of stepwise and orchestrated synthesis,
dendrimers have attracted the attention of the drug-delivery
community due in part to the belief that exquisite control over
composition can counter challenges to absorption, distribution,
metabolism, excretion, and toxicity.1 The field is driven by the
belief that polymeric therapeutics will contribute significantly
to human health by increasing the efficacy of currently used
drugs as well as providing opportunities for the use of new
agents currently precluded from the clinic due to challenges
including low solubility and systemic toxicity.2 This desired
increase in activity could be derived from a number of effects
including prolonged circulation times, reduced toxicity of drugs,
increased drug solubility, and perhaps most importantly, selec-
tive delivery to tumors through either active targeting with
appended ligands for tumor-specific receptors or passive target-
ing that results from the enhanced permeability and retention
(EPR) effect.3
melamine have acute toxicities at dosages ip of 40 mg/kg.
Mortality is observed at 160 mg/kg.5,6b In addition, these cationic
polymers are hemolytic. Here, we use synthesis to explore these
toxicities as a function of surface group with the goal of
identifying a candidate for iv administration. Studies of the in
vitro cytotoxicity and hemolytic potential of six derivatives of
1 (Chart 1, Table 1), including two polycations, three polyanions,
and a PEGylated species, are complemented with acute toxicity
studies of 7 in mice.
Results and Discussion
The dendrimers examined comprise triazines linked by
diamines. While we have reported extensively on these archi-
tectures, 1 and its derivatives (2-7) are unique in a number of
respects. These dendrimers incorporate AB4 terminal groups
providing 48 surface sites that increase the steric bulk on the
periphery. The minimum-energy structures (Figure 1) that result
from gas-phase simulations of 2 and 7 show that the core of
the dendrimer is confined to the center of globular architectures
having diameters of 2.4 and 4.6 nm, respectively. The sizes of
these architectures in aqueous solution, especially that of 7, are
likely larger.
Our previous studies have established that dendrimers based
on melamine are tractable, engineerable targets4 that solubilize
hydrophobic drugs.5 Solubilization does not preclude drug
action.5 These dendrimers attenuate the toxicity of approved
anticancer drugs (methotrexate and 6-mercaptopurine) in vivo,
† Texas A&M University.
(4) (a) Zhang, W.; Tichy, S. E.; Pe´rez, L. M.; Maria, G.; Lindahl, P. A.;
Simanek, E. E. J. Am. Chem. Soc. 2003, 125, 5086. (b) Zhang, W.; Nowlan,
D. T., III; Thomson, L. M.; Lackowski, W. M.; Simanek, E. E. J. Am.
Chem. Soc. 2001, 123, 8914. (c) Steffensen, M. B.; Simanek, E. E. Org.
Lett. 2003, 5, 2359. (d) Zhang, W.; Gonzalez, S. O.; Simanek, E. E.
Macromolecules 2002, 35, 9015.
‡ Texas A&M University Health Science Center.
(1) (a) Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41,
1329. (b) Aulenta, F.; Hayes, W.; Rannard, S. Eur. Polym. J. 2003, 39,
1741. (c) Cloninger, M. J. Curr. Opin. Chem. Biol. 2002, 6, 742. (d) Esfand,
R.; Tomalia, D. A. Drug DiscoVery Today 2001 6, 427-436. (e) Patri, A.
K.; Majoros, I. J.; Baker, J. R., Jr. Curr. Opin. Chem. Biol. 2002, 6, 466.
(2) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347.
(3) (a) Maeda, H. AdV. Enzyme Regul. 2001, 41, 189. (b) Matsumura, Y.;
Maeda, H. Cancer Res. 1986, 6, 6387. (c) Greish, K.; Fang, J.; Inutsuka,
T.; Nagamitsu, A.; Maeda, H. Clin. Pharmacokinet. 2003, 42, 1089.
(5) Zhang,W.; Jiang, J.; Qin, C.; Thomson, L. M.; Parrish, A. R.; Safe, S. H.;
Simanek, E. E. Supramol. Chem. 2003, 15, 607.
(6) (a) Neerman, M. F.; Chen, H. T.; Parrish, A. R.; Simanek, E. E. Unpublished
results. (b) Neerman, M. F.; Zhang, W.; Parrish, A. R.; Simanek, E. E. Int.
J. Pharm. In press.
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J. AM. CHEM. SOC. 2004, 126, 10044-10048
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Dendrimer Candidate Vehicles for Drug Delivery
A R T I C L E S
Chart 1. Dendrimer 1 (R d BOC) in Atomic Detail and 1-7 Shown Schematically with R Identified
Table 1. Library of Dendrimers Used in This Study
The synthesis is convergent in the classical sense (Scheme
1).7 Using BOC-ON, the primary amines of 3,3′-iminobispro-
pylamine are protected to yield 8. Reaction with cyanuric
chloride produces monochlorotriazine 9, which can undergo
chemoselective reaction with the secondary amine of amino-
methylpiperidine to yield 10. Reaction with cyanuric chloride
provides the monochlorotriazine that is ultimately attached to
the hexavalent core. The core is available by reaction of cyanuric
chloride with BOC-piperazine. Upon deprotection of 12,
triamine 13 is reacted with the protected monochlorotriazine
14, a material available in one step in 97% yield. The protected
hypercore 15 is reacted with trifluoroacetic acid before the
ultimate coupling step.
second generation dendron is available in four synthetic steps
necessitating two chromatographic purifications in 75% overall
yield. Thus, the synthesis can been executed in five linear steps
to yield 5 g of material in 56% overall yield and requires one
final chromatographic step to yield pure 1 (>95%). A single
peak observed by HPLC analysis corroborates this conclusion.
The efficiency of the final multicomponent coupling step is
highly dependent on the base used. A survey of diisopropyl-
ethylamine, K2CO3, MTBD,8 BEMP,9 and resin-supported
TBD10 and BEMP11 reveals that resin-supported BEMP provides
the greatest yield and fewest side products.
(7) For a detailed description of dendrimer syntheses, including this hypercore
approach, see: Grayson, S. M.; Fre´chet, J. M. J. Chem. ReV. 2001, 101,
3819.
The last step in the synthesis of 1 involves the coupling of
six second generation monochlorotriazine dendrons, 11, to a
core displaying six reactive piperazine groups, 16. The core of
the dendrimer is available in four synthetic steps necessitating
two chromatographic purifications in 76% overall yield. The
(8) MTBD ) 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene. Schwesinger, R.
Chimia 1985, 39, 269.
(9) BEMP ) 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-
diazaphosphorine. (a) Schwesinger, R. Schlemper, H. Angew. Chem., Int.
Ed. Engl. 1987, 26, 1167. (b) O’Donnell, M. J.; Delgado, F.; Hostettler,
C.; Schwesinger, R. Tetrahedron Lett. 1998, 39, 8775.
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A R T I C L E S
Chen et al.
Figure 1. Gas-phase simulations of 2 and 7 show globular structures with diameters of 2.4 and 4.6 nm, respectively. The dendrimers are shown in blue
(nitrogen) and gray (carbon) with the central triazine in maroon. Surface groups are shown as red (AB4 amines) or orange (PEG2000).
Scheme 1. Synthesis of 1
Dendrimers 2-7 were prepared by quantitatively deprotecting
1 with trifluoroacetic acid before subjecting the resulting
material, 2, to a range of reaction conditions (Table 1) to provide
the small library of dendrimers. These manipulations sacrifice
monodispersity of the resulting dendrimer populations in order
to efficiently address the biological challenges. Monodisperse
populations will be prepared when in vivo tumor-inhibition
activity warrants this action. The table provides the calculated
molecular ions for the indicated number of surface reactions
(10) TBD ) 1,5,7-triazabicyclo[4.4.0]dec-5-ene. (a) Xu, W.; Mohan, R.;
Morrissey, M. M. Tetrahedron Lett. 1997, 38, 7337. (b) Weidner, J. J.;
Parlow, J. J.; Flynn, D. L. Tetrahedron Lett. 1999, 40, 239.
(11) Shuttleworth, S. J.; Nasturica, D.; Gervais, C.; Siddiqui, M. A.; Rando, R.
F.; Lee, N. Bioorg. Med. Chem. Lett. 2000, 10, 2501.
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Dendrimer Candidate Vehicles for Drug Delivery
A R T I C L E S
Figure 3. Cell viability measured using the MTT assays at concentrations
that increase from left to right: 0.001, 0.01, 0.1, 1, and 10 mg/mL.
All the molecules show a concentration-dependent and time-
dependent hemolysis. Hemolysis is more pronounced in cationic
dendrimers than in anionic dendrimers. The PEGylated den-
drimer is the least hemolytic in these experiments.
This trend in hemolytic potential is mirrored in cell viability
assays with Clone 9 cells (Figure 3) using the MTT spectro-
photometric assay.20,21 The cationic dendrimers (2 and 3)
displayed significant cytotoxicity, even at low concentrations.
Anionic surface modification (4-6) afforded protection against
cytotoxicity; however, decreases in viability were observed at
higher concentrations. The PEG-modified dendrimer 7 showed
little cytotoxicity over the range of concentrations surveyed.
We conclude from these experiments that the modified den-
drimers are less toxic to normal cells than is 2.
Microscopic evaluation at 32× of Clone 9 cells after exposure
to unmodified and modified dendrimers bears out the spectro-
photometric assays of viability. Control cells (Figure 4, panel
a) were compared with cells treated with 1 or 10 mg/mL of
cationic dendrimer 2 (panels b and c, respectively) and 1 or 10
mg/mL of the PEGylated dendrimer 7 (panels d and e,
respectively). After 3 h of exposure, both concentrations of 2
induced morphological changes in the cells. Cells treated with
7 appear more similar to untreated cells.
To establish the validity of these in vitro analyses and the
dosage for future tumor inhibition studies, the in vivo toxicity
of 7 was determined. Male C3H mice were administered doses
up to 2.56 g/kg in saline solution by ip injection. No mortality
was observed. After 48 h, serum samples obtained from each
animal showed no significant increases in either urea nitrogen
levels or liver enzyme (alanine transaminase) activity. Accord-
ingly, we conclude that the in vivo toxicity to liver and kidneys
is low enough to warrant further study and that this architecture
is suitable for tumor inhibition studies. Dendrimer 7 was
administered iv in doses up to 1.28 g/kg. Again, serum samples
collected after 24 h showed no significant increases in either
urea nitrogen levels or liver enzyme (alanine transaminase)
activity.
Figure 2. Impact of surface modification on the hemotoxicity of 2-7 at
1 (panel a) and 24 h (panel b). Doses increase for each species from left to
right: 0.001, 0.01, 0.1, 1, and 10 mg/mL.
and the center of the observed distribution of products. On the
basis of analysis of the mass spectra, we conclude that reactions
to produce 3 (with 1H-pyrazole-1-carboxamidine‚HCl)12 and 6
(with succinic anhydride13) proceed almost quantitatively.
Reaction with sultone14 yields 4 with ∼24 of the 48 peripheral
amines functionalized. Reaction of 2 with the vinylphosophonate
diester15 followed by hydrolysis16 leads to formation of tertiary
and quarternary amines. The population is centered at 72
reactions, corresponding to 1.5 reactions for every primary
amine of 2. PEGylation with the NHS ester of a 2000 Da
N-succinimidyl propionate PEG that is terminated with a methyl
group shows the greatest dispersity.17 Molecular weights range
from species with >12 PEG chains (>25%) to dendrimers to
almost fully PEGylated architectures. For these studies, we chose
a mixture whose center of mass corresponds to approximately
half of the 48 surface groups (26) reacting. This mean mass
exceeds the perceived 40 kDa threshold for the EPR effect that
tumors show for polymeric molecules.3 PEGylation can be
driven to completion. Dendrimers 2-7 are soluble in phosphate-
buffered saline at physiological pH.
Red blood cell lysis precludes direct intravenous delivery of
desired agents and often enhances the toxicity of these agents
when administered by other routes. Due to the number of groups,
the periphery of a dendrimer can greatly influence the physical
and biological properties, and accordingly, by changing these
groups, hemolysis can be attenuated. The results of these studies
appear in Figure 2. We examined the hemolytic potential of a
dextran control and 2-7 over a 103 concentration range.18,19
Hemolysis was recorded spectrophotometrically at 1 and 24 h.
Conclusions
A library of six dendrimers that vary in surface chemistry
was prepared from a common precursor available in five steps
in 56% overall yield. The in vitro evaluation of cytotoxicity
and hemolytic potential of this library identified a candidate, 7,
for further in vivo toxicology studies. No toxicity of this
polyPEGylated architecture was observed in mice at doses up
to 2.56 g/kg ip or 1.28 g/kg iv based on blood chemistry analysis
(12) As per Larsen, S. D.; Connell, M. A.; Cudahy, M. M.; Evans, B. R.; May,
P. D.; Meglasson, M. D.; O’Sullivan, T. J.; Schostarez, H. J.; Sih, J. C.;
Stevens, F. C.; Tanis, S. P.; Tegley, C. M.; Tucker, J. A.; Vaillancourt, V.
A.; Vidmar, T. J.; Watt, W.; Yu, J. H. J. Med. Chem. 2001, 44, 1217.
(13) As per Trester-Zedlitz, M.; Kamada, K.; Burley, S. K.; Fenyo¨, D.; Chait,
B. T.; Muir, T. W. J. Am. Chem. Soc. 2003, 125, 2416.
(14) As per Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Pan, Y.; Rege, S. J.
Org. Chem. 1998, 63, 5636.
(15) Rosowsky, A.; Forsch, R. A.; Moran, R. G.; Kohler, W.; Freisheim, J. H.
J. Med. Chem. 1988, 31, 1326.
(16) Palacios, F.; Aparicio, D.; Vicario, J. Eur. J. Org. Chem. 2002, 24, 4131.
(17) As per Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T. Bioconjugate
Chem. 2000, 11, 910.
(18) Carren˜o-Go´mez, B.; Duncan, R. Int. J. Pharm. 1997, 148, 231.
(19) Fischer, D.; Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials
2003, 24, 1121.
(20) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
(21) (a) Sgouras, D.; Duncan, R. J. Mater. Sci.: Mater. Med. 1990, 1, 61. (b)
Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey, H.;
Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. J. Controlled Release
2000, 65, 133.
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Chen et al.
Figure 4. Clone 9 cells grown to confluence (panel a) undergo morphological changes in the presence of 1 mg/mL of 2 (panel b) or 10 mg/mL of 2 (panel
c) but are relatively unaffected by similar concentrations of 7 (panels d and e).
of blood urea nitrogen levels (kidney toxicity) or alanine
transaminase activity (liver toxicity). It is important to note that
units of mg/mL are used throughout these experiments as is
tradition in the toxicology literature. The molecular weights of
dendrimers 2-6 are approximately the same, ranging from 7
to 12 kDa. Dendrimer 7 is significantly larger, with a molecular
weight of ∼60 kDa. Comparisons of hemolysis and cell viability
on a mole:mole basis suggest that 7 is much more similar to
4-6 than casual inspection would suggest. Our choice of
limiting the acute in vivo studies to only the PEGylated
dendrimer 7 (instead of including 4-6) was based primarily
on reports that anionic polymers have anti-coagulant activity
and can stimulate cytokine release.2 The acute toxicity levels
observed for 7 are similar to those observed by Fre´chet and
co-workers in a three-arm poly(ethylene oxide) star polymer,
bearing generation two polyester dendrons with a molecular
weight of 23.5 kDa.22 It should be noted that our toxicity studies
are the result of a single (acute) injection. The chronic toxicity
of the species has not been established and may be different
than the acute toxicity as a result of dendrimer metabolism and
toxicity of the degraded products. Accordingly, these issues, as
well as the biodistribution and excretion of candidate 7 and
similar architectures, will be pursued in due course to satisfy
our aim of rationally approaching in vivo tumor assays.
Acknowledgment. This work was supported by the NIH
(NIGMS 65450) and the Center for Microencapsulation and
Drug Delivery at Texas A&M University. Dr. Paul Brandt
(Medical Pharmacology and Toxicology, TAMU HSC) is
thanked for access to his microscope.
Supporting Information Available: Complete description of
biological, computational, and synthetic methods. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA048548J
(22) Padilla De Jesus, O. L.; Ihre, H. R.; Gagne, L.; Fre´chet, J. M. J.; Szoka, F.
C., Jr. Bioconjugate Chem. 2002, 13, 453.
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