Low toxic, thermoresponsive dendrimers based on oligoethylene glycols with sharp and fully reversible phase transitions

Article abstract of DOI:10.1039/b814192d

Novel first (G1) and second (G2) generation dendrimers based on three-fold branched oligoethylene glycol dendrons are efficiently synthesized which show characteristic thermoresponsive behavior and negligible cytotoxicity (for G2). The Royal Society of Ch

Full text of DOI:10.1039/b814192d

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Low toxic, thermoresponsive dendrimers based on oligoethylene glycols
with sharp and fully reversible phase transitionsw
Wen Li,a Afang Zhang,*ab Yong Chen,c Kirill Feldman,a Hua Wud and
A. Dieter Schlu
¨
ter*a
Received (in Cambridge, UK) 18th August 2008, Accepted 4th September 2008
First published as an Advance Article on the web 9th October 2008
DOI: 10.1039/b814192d
Novel first (G1) and second (G2) generation dendrimers based on
three-fold branched oligoethylene glycol dendrons are efficiently
synthesized which show characteristic thermoresponsive behavior
and negligible cytotoxicity (for G2).
hyperbranched polymers with such units,12 hoping that this
surface decoration would render the products also water-
soluble, non-toxic and bio-compatible.13 OEG and PEG are
thermoresponsive. However, their LCSTs are in the range of
100–176 1C, which is rather high for many bio-related applica-
tions.14 This may perhaps be the reason why the thermo-
responsive behavior of the decorated dendrimers was not
investigated until recently, when Dai and Chang reported
the thermoresponsiveness of G1 dendrimers. They however
did not give information on the reversibility of their thermal
transitions.15 It was discovered that dendronized polymers
with specifically designed OEG-based dendrons exhibit
unprecedentedly sharp, reversible and fast transitions in
aqueous solution with LCSTs in the attractive range of
27–65 1C depending on the particular structure.16 In order
to find out whether this rather unique behavior is associated
with the structure of dendronized polymers, in which pendent
dendrons are forced into a close packing by the backbone, or
with that of the dendrons themselves, we sought for simpler
and even easier to access macromolecules carrying exactly
these OEG-based dendrons. We here report on the synthesis
of dendrimers Me-G1, Et-G1, Me-G2, and Et-G2 (Fig. 1),
which contain these dendrons as integral parts. These novel
dendrimers’ thermal transitions were studied in some detail by
turbidity measurements. The aggregates that formed upon
collapse above the LCST were characterized using optical
and atomic force microscopy (AFM). Having such interesting
compounds at hand, their cytotoxicity to both B16F1 and
HaCat cell lines was also investigated and compared to linear
A large variety of dendrimers1 with different chemical struc-
tures have been developed in the last decades for applications
in areas such as biomaterials, molecular devices, and cataly-
sis.2 Application-driven research also included the synthesis of
representatives which are responsive to chemical or physical
stimuli, such as light,3 solvent,4 and pH.5 Attention has further
been paid to thermally responsive dendrimers.6 For example,
in their pioneering work Kono and co-workers7 reported a
series of PAMAM dendrimers which were rendered thermo-
responsive (with lower critical solution temperature, LCST, in
the range of 16–80 1C) by attaching short thermoresponsive
units to the periphery. In a related approach, You, Hong
et al.8 presented dendrimers with peripherally grafted long
poly(N-isopropylacrylamide) (PNiPAM) chains. For applica-
tion purposes it would be desirable to have materials with
sharp, fast, and fully reversible phase transitions covering a
broad temperature range.9 Unfortunately, these conditions are
only partially met by the above cases. Either there is no mention
of whether or not the transitions are reversible7 or the transi-
tions take place in a relatively broad temperature range of
B5 1C.8 Also it seems that upon increased heating above the
LCST, aggregates can disassemble.7 The cytotoxicity of
thermally responsive dendrimers has not yet been investigated
in great detail. It should be mentioned however that, for
example, PAMAM exhibits considerable cytotoxicity.10
Linear oligo- and polyethylene glycols (OEG, PEG) are
water-soluble macromolecules that exhibit low toxicity and
excellent biocompatibility.11 This motivated us to decorate
dendritic structures such as dendrons, dendrimers, and
a Institute of Polymers, Department of Materials, ETH Zurich,
Wolfgang-Pauli-Strasse 10, HCI G525, 8093 Zurich, Switzerland.
E-mail: zhang@mat.ethz.ch; E-mail: ads@mat.ethz.ch;
Fax: (+41) 44-6331390
b School of Materials Science and Engineering, Zhengzhou University,
Daxue Beilu 75, Zhengzhou, 450052, China
c Institute of Pharmaceutical Sciences, ETH Zurich,
Wolfgang-Pauli-Strasse 10, HCI H303, 8093 Zurich, Switzerland
¨
d Institute for Chemical and Bioengineering, ETH Zurich,
Wolfgang-Pauli-Str. 10, HCI F139, 8093 Zurich, Switzerland
w Electronic supplementary information (ESI) available: Experimental
procedures and details; NMR spectra of new compounds and
polymers; optical microscopy movie. See DOI: 10.1039/b814192d
Fig. 1 Chemical structures of the dendrimers Me-G1, Et-G1, Me-G2
and Et-G2 discussed in this study.
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Fig. 2 (a) Plots of transmittance vs. temperature (500 nm, 0.2 1C minÀ1
;
solid line: heating; dashed line: cooling) for 0.25 wt% aqueous
solutions of dendrimers. (b) Dependence of LCSTs on concentrations
of dendrimers and related polymers. Detailed plots of transmittance
vs. temperature are shown in Fig. S1 in the ESI.w
amounts of aggregates below the LCST, while the aggregates
formed above are large and compact enough to scatter light
efficiently. (2) All of the dendrimers investigated show sharp
transitions in both heating and cooling directions (o1 1C),
whereby those of the ethoxy-terminated dendrimers (o0.8 1C)
are even sharper than methoxy-terminated ones (o11). (3)
Very small hystereses are observed between heating and
cooling, which suggests sensitive and fully reversible hydration
and dehydration processes. (4) The LCSTs are tunable in the
range of 27–65 1C which includes the highly relevant transition
of Et-G2 at 36 1C. These different temperatures reflect the
differences in hydrophilicity. It is reasonable to assume that
the second generation and methoxy terminated dendrimers are
more hydrophilic than the first generation and ethoxy termi-
nated ones, respectively.16 The concentration dependence of
the LCST was also investigated in the range of 0.05–2.0 wt%
and the results are plotted in Fig. 2b. Obviously, the lower the
dendrimer concentration, the higher the LCST and the
broader the phase transition (see Fig. S1 in the ESIw). This
may be related to an aggregation kinetic effect, i.e., the lower
the dendrimer concentration, the longer the time needed for
the formation of aggregates. If one compares the thermo-
responsive behavior of the present dendrimers with the
reported dendronized polymers Me-PG1 and Me-PG2,16 two
differences become evident: Both the concentration and
generation dependence of the LCST are lower for the latter
than for the former.
Scheme 1 Synthesis procedures for dendrimers Et-G1, Me-G1, Et-G2
and Me-G2. Reagents and conditions: (a) SOCl2, DMAP, DCM, rt,
4 h (72–84%); (b) THPE, K2CO3, KI, DMF, 80 1C, 24 h (88–90%); (c)
THPE, Cs2CO3, KI, DMF, 80 1C, 48 h (73–74%).
PEG in order to assess their potential applicability in the
biomedical context.
The synthesis procedures for the dendrimers are outlined in
Scheme 1. Starting from the known G1 and G2 dendron
alcohols 1a, 1c, 2a and 2c,16 reaction with SOCl2 in the
presence of 4-dimethylaminopyridine (DMAP) afforded the
corresponding benzyl chlorides 1b, 1d, 2b and 2d, respectively.
The dendrimers were synthesized via Williamson etherification
of corresponding dendron benzyl chlorides with the core
molecule 1,1,1-tris(4-hydroxyphenyl)ethane, THPE. Et-G1
and Me-G1 were obtained with high yields (88–90%) by the
reaction of 1b and 1d with THPE in the presence of K2CO3
and KI with the dendrons in an excess of 10% per phenolic
function. Application of the same method furnished dendri-
mers Me-G2 and Et-G2 in very low yields only (B5%).
Compounds in which THPE had reacted with one or two
dendrons were the main products. Therefore, optimized con-
ditions were applied: Cs2CO3 was used instead of K2CO3 and
a larger excess (50%) of 2b or 2d applied. Additionally the
reaction time was extended from 24 h to 48 h. Eventually, both
Me-G2 and Et-G2 were obtained in yields of 73–74%. All new
1
compounds were characterized by H and 13C NMR spectro-
The aggregation process of dendrimers in aqueous solution
was followed by dynamic light scattering (DLS). Large ag-
gregates with sizes in the range of 2–5 micrometres were
observed above the LCST for all G1 and G2 dendrimers (see
Fig. S2 in the ESIw). The aggregates were also directly
observed in aqueous solutions with optical microscopy. As
shown in Fig. 3a, Et-G2 formed spherical aggregates with sizes
around 2–4 micrometres, which is in good agreement with the
results from DLS. Its thermally induced aggregation and
segregation process followed by optical microscopy (OM)
was video-taped in time intervals of one second (for the movie,
see the ESIw). As shown in the movie, below the LCST, no
aggregates were visible and above, large spherical aggregates
appeared suddenly, and their size remained nearly unchanged.
They disappeared again when the solution temperature de-
creased below the LCST. This process was repeated numerous
times and is fully reversible. The aggregates were also observed
scopy, high resolution MALDI-TOF mass spectrometry, as
well as elemental analysis to guarantee high purities. All
dendrimers gave a single spot on TLC.
By visual inspection all dendrimers are water-soluble at
room temperature; the clear aqueous solutions turn turbid
however when heated. The temperatures at which these transi-
tions occur (commonly, though not precisely referred to as
LCST) and their course were investigated by turbidity mea-
surements using UV/vis spectroscopy. The transmittance dur-
ing the heating and cooling processes of aqueous solutions
from Et-G1, Et-G2, Me-G1 and Me-G2 are plotted in Fig. 2a.
The respective LCSTs are 27, 36, 51, and 65 1C. From these
transition curves, the following conclusions can be drawn: (1)
The transmittance below the LCST is close to 100%, while
that above is close to zero. This suggests that for the concen-
trations used, the dendrimer solutions do not contain sizeable
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dendrimers the cell viability was around 100% up to a con-
centration of 100 mg mLÀ1. These latter results compare
favorably with linear PEG. Unlike the G1 dendrimers, the
cell morphologies after exposure to the G2 dendrimer solu-
tions remained virtually unchanged as compared with these of
the control cells (Fig. S3 in the ESIw). Both results indicate a
rather low toxicity of the G2 dendrimers.
Profs. M. Detmar and P. Walde (ETH Zurich) are thanked
for their support with cytotoxicity and AFM measurements,
respectively. Prof. A. Vasella (ETH Zurich) is thanked for
kindly providing access to his instruments. This work was
supported by ETH Research Grants (ETH-1608-1 and
ETH-0908-2). A. Z. thanks for the financial support from
the National Natural Science Foundation of China (Nos.
20374047 and 20574062).
Fig. 3 (a) Optical micrograph of the aggregates in 0.25 wt% aqueous
solutions of Et-G2. Note that the white features are the ones in focus.
The inset: four-times enlarged feature. (b) Tapping mode AFM image
of the aggregates on HOPG from 0.25 wt% aqueous solutions of
Et-G2. (c) Cross-sectional profile of AFM image in (b).
Notes and references
1 (a) S. M. Grayson and J. M. J. Frechet, Chem. Rev., 2001, 101,
´
3819; (b) B. Helms and E. W. Meijer, Science, 2006, 311, 929.
2 (a) R. Van Heerbeek, P. C. J. Kamer, P. W. N. M. Van Leeuwen
and J. N. H. Reek, Chem. Rev., 2002, 102, 3717; (b) M. E. Van der
Boom, Angew. Chem., Int. Ed., 2002, 41, 3363; (c) S.-E. Stiriba, H.
Frey and R. Haag, Angew. Chem., Int. Ed., 2002, 41, 1329; (d) C. C.
Lee, J. A. MacKay, J. M. J. Frechet and F. C. Szoka, Nat.
´
Biotechnol., 2005, 23, 1517; (e) L. E. Euliss, J. A. DuPont, S.
Gratton and J. DeSimone, Chem. Soc. Rev., 2006, 35, 1095; (f) D.
Schaffert and E. Wagner, Gene Ther., 2008, 15, 1.
3 (a) J. W. Weener and E. W. Meijer, Adv. Mater., 2000, 12, 741; (b)
F. Puntoriero, P. Ceroni, V. Balzani, G. Bergamini and F. Vogtle,
¨
J. Am. Chem. Soc., 2007, 129, 10714.
4 (a) I. Gitsov and J. M. J. Frechet, J. Am. Chem. Soc., 1996, 118,
´
3785; (b) A. L. Hofacker and J. R. Parquette, Angew. Chem., Int.
Ed., 2005, 44, 1053.
5 (a) G. R. Newkome, J. K. Young, G. R. Baker, R. L. Potter, L.
Audoly, D. Cooper and C. D. Weis, Macromolecules, 1993, 26,
2394; (b) X. S. Feng, D. Taton, R. Borsali, E. L. Chaikof and Y.
Gnanou, J. Am. Chem. Soc., 2006, 128, 11551.
Fig. 4 Cytotoxicity of dendrimers to B16F1 (a) and HaCat cells (b)
by CCK-8 assay. Data are mean values plus/minus standard deviation
of four samples/cultures.
in dry state on solid substrates (HOPG) by AFM. For sample
preparation, an aqueous solution of Et-G2 was pre-heated
above its LCST (B50 1C) and then spin-coated (2000 rpm)
onto the pre-heated HOPG substrate. Fig. 3b shows the
tapping-mode image of the aggregates, which very much like
in OM appeared spherical. The cross-sectional profile (Fig. 3c)
indicates the aggregates on the substrate with an average size
of roughly around 30 nm (height) Â1 mm (width), which
suggests the aggregates in dry state to be smaller than those
observed in aqueous solution. Further studies are on the way
to elucidate the mechanism of aggregate formation aiming,
besides others, at an understanding of the origin of this unique
behavior.
6 (a) M. C. Parrott, E. B. Marchington, J. F. Valliant and A.
Adronov, J. Am. Chem. Soc., 2005, 127, 12081; (b) Y. Zhou, D.
Yan, W. Dong and Y. Tian, J. Phys. Chem. B, 2007, 111, 1262;
(c) H. Lee, J. A. Lee, Z. Poon and P. T. Hammond, Chem.
Commun., 2008, 3726.
7 (a) Y. Haba, A. Harada, T. Takagishi and K. Kono, J. Am. Chem.
Soc., 2004, 126, 12760; (b) Y. Haba, C. Kojima, A. Harada and K.
Kono, Macromolecules, 2006, 39, 7451.
8 Y. Z. You, C. Y. Hong, C. Y. Pan and P. H. Wang, Adv. Mater.,
2004, 16, 1953.
9 W. T. S. Huck, Mater. Today, 2008, 11, 24.
10 R. Duncan and L. Izzo, Adv. Drug Delivery Rev., 2005, 57, 2215.
11 M. S. Thompson, T. P. Vadala, M. L. Vadala, Y. Lin and J. S.
Riffle, Polymer, 2008, 49, 345.
12 (a) J.-G. Li, C. Meng, X.-Q. Zhang, L. Zhang and A. Zhang, Prog.
Chem., 2006, 18, 1157; (b) V. Gajbhiye, P. V. Kumar, R. K. Tekade
and N. K. Jain, Curr. Pharm. Des., 2007, 13, 415.
13 (a) N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H.
Frey, J. W. Weener, E. W. Meijer, W. Paulus and R. Duncan, J.
Controlled Release, 2000, 65, 133; (b) H.-T. Chen, M. F. Neerman,
A. R. Parrish and E. E. Simanek, J. Am. Chem. Soc., 2004, 126,
10044.
Because of the rather attractive thermoresponsive charac-
teristics of the dendrimers reported here, their cytotoxicity17
was investigated using B16F1 and HaCat cell lines, and the
results were compared with linear PEG (Mn = 5600). These
cell lines were incubated with aqueous solutions of all den-
drimers (concentrations: 1–100 mg mLÀ1) for 48 h at 37 1C and
treated with a CCK-8 assay for another 3 h. The cytotoxicity
results are plotted in Fig. 4. The cell viability fell to o50% for
B16F1 and o80% for HaCat, respectively, for both G1
dendrimers (above concentration 50 mg mLÀ1), with Et-G1
being slightly more toxic than Me-G1. In contrast, for both G2
14 S. Saeki, N. Kuwahara, M. Nakata and M. Kaneko, Polymer,
1976, 17, 685.
15 D. W. Chang and L. Dai, J. Mater. Chem., 2007, 17, 364.
16 (a) W. Li, A. Zhang, K. Feldman, P. Walde and A. D. Schluter,
¨
Macromolecules, 2008, 41, 3659; (b) W. Li, A. Zhang and A. D.
Schluter, Chem. Commun., 2008, DOI: 10.1039/b811464a.
¨
17 For cytotoxicity of dendrimers, see for example: U. Boas and P. M.
H. Heegaard, Chem. Soc. Rev., 2004, 33, 43.
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