Fluorescence and aggregation behavior of poly(amidoamine) dendrimers peripherally modified with aromatic chromophores: The effect of dendritic architectures

Article abstract of DOI:10.1021/ja048219r

PAMAM dendrimers of the zeroth to fifth generation (G0-5) have been peripherally modified with phenyl, naphthyl, pyrenyl, and dansyl chromophores. Their fluorescence behaviors are strongly affected by the dendritic architectures at different generations.

Full text of DOI:10.1021/ja048219r

Published on Web 10/29/2004
Fluorescence and Aggregation Behavior of Poly(amidoamine)
Dendrimers Peripherally Modified with Aromatic
Chromophores: the Effect of Dendritic Architectures
†
†
,†
†
†
†
Bing-Bing Wang, Xin Zhang, Xin-Ru Jia,* Zi-Chen Li, Yan Ji, Ling Yang, and
Yen Wei*,‡
Contribution from the Department of Polymer Science and Engineering, College of Chemistry
and Molecular Engineering, Peking UniVersity, Beijing 100871, China, and Department of
Chemistry, Drexel UniVersity, Philadelphia, PennsylVania 19104
Received March 29, 2004; E-mail: xrjia@chem.pku.edu.cn; weiyen@drexel.edu
Abstract: PAMAM dendrimers of the zeroth to fifth generation (G0-5) have been peripherally modified
with phenyl, naphthyl, pyrenyl, and dansyl chromophores. Their fluorescence behaviors are strongly affected
by the dendritic architectures at different generations. These dendrimers modified with hydrophobic
chromphores can self-organize into vesicular aggregates at the low generations G0-3 in water. The size
and aggregation number of these vesicles decrease with increasing generation from G0 to G3. Critical
aggregation concentration determined by fluorescence spectroscopy reveals that these aggregates can
be favorably formed in the order of G3 > G2 > G1. In contrast to the vesicles made from traditional
amphiphilic compounds, these dendrimer-based vesicles are very adhesive due to the H-bonding interaction
and entanglement of dendritic branches located in the outer layer. A large number of multivesicle assemblies,
i.e., “twins” and “quins” consisting of two and five vesicles, were clearly identifiable with transmission electron
(
TEM) and atomic force microscopy. For the dendrimers with peripheral pyrenyl chromophores, triangle-
like vesicles were observed in water. The hydrophobic interphase thickness of the vesicular bilayer is ca.
.0-3.2 nm determined by fluorescence resonance energy transfer methods, which agrees well with the
2
thickness directly observed with TEM.
6
7
8
9
Introduction
azobenzene, dansyl sulfonate, glucose, porphyrins, cyano-
1
0
11
biphenyl mesogens, oligo(p-phenylene vinylene), aromatic
1
2
Since the pioneering work by Tomalia and V o¨ gtle about
0 years ago, we have witnessed an explosive growth of a new
amines, and cumarin.13 The peripheral modification of den-
drimers with photo- and/or electroactive moieties generally
resulted in interesting specific functions. Compared to their
linear analogues, the unusual molecular architecture of dendritic
polymers provides a suitable framework to achieve unique
photochemical and photophysical properties. As examples,
12
2
class of highly symmetric and branched, monodisperse, aestheti-
cally appealing macromolecules with well-defined shape and
size, i.e., dendrimers. Recent research emphasis seems to shift
from the synthesis of novel dendrimers to their properties and
potential applications. Modification and functionalization of
3
4
dendritic periphery are of particular keys to tune and manipulate
the properties of dendrimers such as solubility and viscosity as
well as photochemical and photophysical behavior. The average
spacing between peripheral chromophores can be controlled by
varying the dendrimer generations.
A large number of papers have reported on the peripheral
modification of dendrimers with various moieties such as
(
6) Tsuda, K.; Dol, G. C.; Gensch, T.; Hofkens, J.; Latterini, L.; Weener, J.
W.; Meijer, E. W.; de Schryver, F. C. J. Am. Chem. Soc. 2000, 122, 3445-
3452.
5
(
7) (a) Vicinelli, V.; Ceroni, P.; Maestri, M.; Balzani, V.; Gorka, M.; Vogtle,
F. J. Am. Chem. Soc. 2002, 124, 6461-6468. (b) Teobaldi, G.; Zerbetto,
F. J. Am. Chem. Soc. 2003, 125, 7388-7393. (c) Balzani, V.; Ceroni, P.;
Gestermann, S.; Kauffmann, C.; Gorka, M.; Vogtle, F. Chem. Commun.
2000, 122, 853-854.
(
8) Schmitzer, A.; Perez, E.; Rico-Lattes, I.; Lattes, A.; Rosca, S. Langmuir
1999, 15, 4397-4403.
(9) Yeow, E. K. L.; Ghiggino, K. P.; Reek, J. N. H.; Crossley, M. J.; Bosman,
A. W.; Schenning, A. P. H. J.; Meijer, E. W. J. Phys. Chem. B 2000, 104,
†
Peking University.
Drexel University.
2596-2606.
‡
(10) (a) Ponomarenko, S. A.; Boiko, N. I.; Shibaev, V. P.; Richardson, R. M.;
Whitehouse, I. J.; Rebrov, E. A.; Muzafarov, A. M. Macromolecules 2000,
33, 5549-5558. (b) Baars, M. W. P. L.; Sontjens, S. H. M.; Fischer, H.
M.; Peerlings, H. W. I.; Meijer, E. W. Chem.sEur. J. 1998, 4, 2456-
2466.
(11) Schenning, A. P. H. J.; Peeters, E.; Meijer, E. W. J. Am. Chem. Soc. 2000,
122, 4489-4495.
(12) Freeman, A. W.; Koene, S. C.; Malenfant, P. R. L.; Thompson, M. E.;
Frechet, J. M. J. J. Am. Chem. Soc. 2000, 122, 12385-12386.
(13) (a) Adronov, A.; Gilat, S. L.; Frechet, J. M. J.; Ohta, K.; Neuwahl, F. V.
R.; Fleming, G. R. J. Am. Chem. Soc. 2000, 122, 1175-1185. (b) Gilat, S.
L.; Adronov, A.; Frechet, J. M. J. Angew. Chem., Int. Ed. 1999, 38, 1422-
1427.
(
1) Tomalia, D. A.; Baker, H.; Dewald, J. R.; Hall, M.; Kallos, G.; Martin, S.;
Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117-132.
(
(
2) Buhleier, E. W.; Wehner, W.; V o¨ gtle, F. Synthesis 1978, 155-158.
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Concepts, Syntheses and PerspectiVes; VCH: Weinheim, Germany, 1996.
(
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4) For selected reviews: (a) Sugiura, K. Top. Curr. Chem. 2003, 228, 65-
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(
15180
9
J. AM. CHEM. SOC. 2004, 126, 15180-15194
10.1021/ja048219r CCC: $27.50 © 2004 American Chemical Society

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
Scheme 1. Peripheral Modification of PAMAM Dendrimers with Phenyl, Naphthyl, Pyrenyl, and Dansyl Chromophoresa
a
Reagents and conditions: (i) methanol, Na2SO4, 60 °C; (ii) methanol, 30 °C, NaOH; (iii-v) acetone/methanol (4:1 v/v), room temperature, 24 h.
Crooks and Baker14 prepared the poly(propylene imine) den-
drimers functionalized with pyrene chromophores and studied
the correlation of spectroscopic properties with dendrimer
disklike shape to cylindrical columns by the peripheral modi-
fication with lipophilic chains. Meijer and co-workers
2
3
6,22
synthesized the poly(propylene imine) dendrimers with hydro-
phobic alkyl chains at their periphery, which form various self-
assemblies from a globular inverted micellar arrangement to a
cylindrical amphoteric structure. As mentioned above, much
work has been devoted to the studies of the amphiphilic
dendrimers bearing long soft chains at their periphery. However,
little attention was paid to the aggregation behavior of the
flexible hydrophilic dendrimers with rigid hydrophobic periph-
ery.
In this article, we describe the peripheral modification of
PAMAM dendrimers with naphthyl, pyrenyl, phenyl, and dansyl
chromophores (Scheme 1). Their spectroscopic behavior has
been explored with the aid of a series of small molecular model
compounds. The aggregation and self-assembly of the hydro-
philic PAMAM dendrimers bearing hydrophobic aromatic
chromophores at their periphery have been observed and
investigated systematically. To the best of our knowledge, this
is the first report on the self-organization of the hydrophilic
dendritic backbone with rigid hydrophobic periphery to form
ordered aggregates.
15a-c
structures. Moore and co-workers
studied the energy
migration in phenylacetylene dendrimers having perylene chro-
mophores. Fox and co-workers1 modified fan-shaped polyether
dendrons with pyrene and naphthalene chromophores and
studied the fluorescence resonance energy transfer between the
5d
5
,16
core and peripheral chromophores. Frechet and co-workers
synthesized a series of dendrimers bearing light-harvesting
chromophores at their periphery, which can transfer outer energy
to a central core. Beyond any doubt, a better understanding of
the photochemical and photophysical behaviors of the peripher-
ally modified dendrimers and their structure-property relation-
ship is necessary and of great significance in order to develop
innovative applications for these unique molecules.
On the other hand, amphiphilic dendrimers have recently
attracted an enormous amount of attention because of their
17
potential applications in molecular encapsulation, drug deliv-
1
8
19
ery, and nanoscopic transport. The “unimolecular micelles”
20
as termed by Newkome have been made by modifying
dendrimers with long hydrophobic or hydrophilic chains at their
periphery, which can serve as a “dendritic box” for the
Experimental Section
2
1
encapsulation and transport of lipophilic compounds. Percec
2
3
Materials, measurements, synthesis, and characterization of model
compounds, i.e., N-(N′-1-naphthylsuccinimido)isobutylamine (MNp),
N-(N′-1-pyrenylsuccinimido)isobutylamine (MPy), N,N-bis(N′-1-naph-
thylsuccinimido)isobutylamine (BNp), N,N-bis(N′-1-pyrenylsuccinimi-
do)isobutylamine (BPy), were provided in the Supporting Information.
TEM and AFM Measurements. Transmission electron microscopic
and co-workers reported that various self-assemblies can be
formed by using different generation dendrons as building
blocks. The molecular architectures can be designed from a
(
14) Baker, L. A.; Crooks, R. M. Macromolecules 2000, 33, 9034-9039.
15) (a) Kopelman, R.; Shortreed, M.; Shi, Z. Y.; Tan, W. H.; Xu, Z. F.; Moore,
J. S.; BarHaim, A.; Klafter, J. Phys. ReV. Lett. 1997, 78, 1239-1242. (b)
Swallen, S. F.; Zhu, Z. G.; Moore, J. S.; Kopelman, R. J. Phys. Chem. B
(
(TEM) measurement was performed on a JEM-100 CXII microscope,
2
000, 104, 3988-3995. (c) Devadoss, C.; Bharathi, P.; Moore, J. S. J.
Am. Chem. Soc. 1996, 118, 9635-9644. (d) Stewart, G. M.; Fox, M. A. J.
Am. Chem. Soc. 1996, 118, 4354-4360. (e) Devadoss, C.; Bharathi, P.;
Moore, J. S. Macromolecules 1998, 31, 8091-8099.
(22) (a) Schenning, A. P. H. J.; Elissen-Roman, C.; Weener, J. W.; Baars, M.
W. P. L.; van der Gaast, S. J.; Meijer, E. W. J. Am. Chem. Soc. 1998, 120,
8199-8208. (b) Stevelmans, S.; van Hest, J. C. M.; Jansen, J. F. G. A.;
van Boxtel, D. A. F. J.; de Brabander-van den Berg, E. M. M.; Meijer, E.
W. J. Am. Chem. Soc. 1996, 118, 7398. (c) Bosman, A. W.; Janssen, H.
M.; Meijer, E. W. Chem. ReV. 1999, 99, 1665-1688.
(23) (a) Percec, V.; Glodde, M.; Bera, T. K.; Miura, Y.; Shiyanovskaya, I.;
Singer, K. D.; Balagurusamy, V. S. K.; Heiney, P. A.; Schnell, I.; Rapp,
A.; Spiess, H. W.; Hudson, S. D.; Duan, H. Nature 2002, 419, 384-387.
(b) Percec, V.; Ahn, C. H.; Cho, W. D.; Jamieson, A. M.; Kim, J.; Leman,
T.; Schmidt, M.; Gerle, M.; Moller, M.; Prokhorova, S. A.; Sheiko, S. S.;
Cheng, S. Z. D.; Zhang, A.; Ungar, G.; Yeardley, D. J. P. J. Am. Chem.
Soc. 1998, 120, 8619-8631. (c) Hudson, S. D.; Jung, H. T.; Percec, V.;
Cho, W. D.; Johansson, G.; Ungar, G.; Balagurusamy, V. S. K. Science
1997, 278, 449-452.
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002, 124, 11848-11849. (b) Frechet, J. M. J. Proc. Natl. Acad. Sci. U.S.A.
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17) (a) Niu, Y. H.; Crooks, R. M. Chem. Mater. 2003, 15, 3463-3467. (b)
Nierengarten, J. F. Top. Curr. Chem. 2003, 228, 87-110. (c) Zimmerman,
S. C.; Lawless, L. J. Top. Curr. Chem. 2001, 217, 95-120.
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1
(
J. AM. CHEM. SOC.
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VOL. 126, NO. 46, 2004 15181

A R T I C L E S
Wang et al.
operating at an acceleration voltage of 100 kV. For the observation of
size and distribution of vesicle particles, a drop of sample solution
(concentration: 0.5 mg/mL) was placed on carbon-coated Formvar
copper grids. About 2 min after the deposition, the grid was tapped
with filter paper to remove surface water and air-dried. Negative staining
was performed by addition of a drop of 2 wt % solution of uranyl
acetate. An atomic force microscope (AFM, Dimension 3000, Digital
instrument) was used in the tapping mode according to the literature
2
4a
procedures. A drop of sample solution (concentration: 0.5 mg/mL)
was placed on the mica surface. AFM measurement was performed
after the surface water was volatilized naturally at room temperature.
PAMAM Dendrimers G0-5 Peripherally Modified with Phenyl
Groups (PAMAM-Ph G0-5) PAMAM-Ph G1. Benzaldehyde (10
mL, 0.037 g, 3.08 mmol) was added to a methanol solution (50 mL)
of the first generation PAMAM dendrimer G1 (0.50 g, 0.35 mmol) in
the presence of anhydrous sodium sulfate (2.0 g). After the reaction at
Figure 1. Absorption and emission spectra of PAMAM-Py G0-5 and
6
0 °C for 12 h under stirring, the system was cooled to room
mono- and bis-substituted model compounds MPy and BPy in dichlo-
temperature and the solvent was removed under a reduced pressure.
The residue was dissolved again in chloroform (5 mL). The solvent
and excess benzaldehyde were removed under reduced pressure. The
resulting yellow semisolid crude product was purified by repeated
dissolution in methanol (5 mL) and precipitation in anhydrous diethyl
ether (100 mL) with vigorous stirring. The precipitate was collected
-6
romethane. [Py] ) 2.5 × 10 M. The emission spectra are normalized to
ex
E M
the intensity at 378 nm. λ ) 335 nm. Inset: Variation of the pyrene I /I
-
6
ratio with generations in dichloromethane. [Py] ) 2.5 × 10 M.
ring), δ2.763 (56H, 28-CH
12-CH -NH-SO -), δ3.185 (48H, 8-N(CH
CH -CO-). IR (KBr pellet, cm ): 3078, 3480, 3360, 2849, 1647,
1542, 1456, 1318, 1324, 790. 13C NMR (CDCl
, ppm): 174.94, 155.28,
141.74, 132.22, 126.91, 123.17, 112.08, 50.33, 44.46, 35.35. MALDI-
2
-N(-CH
2
-)-CH
2
-CH
2
-)
2
, δ3.481 (24H,
2
2
3 2
)
), δ2.534 (28H, 14-
-
1
and further purified by dialysis using a dialysis membrane (M
to afford final product (0.1 g). Yield: 39%. FT-IR: (KBr pellet, cm
278, 3061, 2936, 2841, 1645, 1552, 1450, 1378, 1254, 757, 720, 695.
W
) 1000)
2
-
1
)
3
3
1
H NMR (CDCl
8H, s, 8-CHdNs), δ3.67 (16H, 8-CH
-NHsCH sCH sNdCs), δ3.18 (8H, 4-NHsCH
sCH s), δ2.65 (22H, 11-CH sN(sCH s)sCH
12H, 6-CH sN(sCH s)sCH s), δ 2.25 (24H, 12-CH
, ppm): 172.49, 162.91, 135.69, 130.83, 128.56, 128.07,
0.24, 50.13, 40.13, 33.95. MALDI-TOF MS (matrix: dithranol/silver
trifluoroacetate )1:1 w/w) calcd for C118 12 [M]: 2132.
3
, TMS, ppm): δ7.36-7.66 (40H, aromatic ring), δ8.25
sCH sNdCHs), δ3.50 (16H,
sCH sN(sCH
sCH s), δ2.46
sCOs). 13
TOF MS (matrix: 2,5-dihydroxybenzoic acid) Calcd for C159
[M]: 3302.4. Found: 3302 (M) .
H N O S
220 34 28 8
+
(
8
)
(
2
2
2
2
2
2
2
s
PAMAM-DNS G0 and G2-5 were synthesized in the same manner
2
2
2
2
2
as PAMAM-DNS G1. Their characterization was provided in the
C
Supporting Information. H NMR spectra indicated that the reaction
1
2
2
2
2
NMR (CDCl
6
3
extent of amino group (-NH ) on the dendrimer surface with dansyl
2
chloride was 90% for G0, 92% for G1, 90% for G2, 86% for G3, 86%
for G4, and 80% for G5.
PAMAM Dendrimers G0-5 Peripherally Modified with Pyrenyl
Chromophores (PAMAM-Py G0-5) PAMAM-Py G1. A methanol
solution (25 mL) of PAMAM G1 (0.0136 g, 0.00952 mmol) was added
dropwise to an acetone solution (30 mL) of N-(1-pyrenyl)maleimide
160 26
H N O
+
+
Found: 2133 (M + H ), 2155 (M + Na ).
PAMAM-Ph G0 and G2-5 were synthesized in the same manner
as PAMAM-Ph G1. Their spectral characterization was provided in
1
the Supporting Information. H NMR spectra indicate that the reaction
2
extent of the amino group (-NH ) on the dendrimer surface with
(0.050 g, 0.168 mmol) at 0 °C. The resultant solution was kept at 30
benzaldehyde was 90% for G0, 94% for G1, 92% for G2, 80% for G3,
°C with stirring for 48 h. After evaporating the solvent under a reduced
8
5.7% for G4, and 88% for G5.
pressure, the residue was dissolved in methanol (5 mL), which was
then dropped into anhydrous diethyl ether (100 mL) with stirring. A
white precipitation was collected by suction filtration, then dissolved
again in methanol (10 mL) and dialyzed with a dialysis bag (MW 3500)
using methanol as a solvent at room temperature for 24 h. After
evaporating methanol under a reduced pressure and drying in a vacuum
PAMAM Dendrimers G0-5 Peripherally Modified with Dansyl
Chromophores (PAMAM-DNS G0-5) PAMAM-DNS G1. A metha-
nol solution (20 mL) of PAMAM G1 (0.10 g, 0.070 mmol) was added
dropwise to the aqueous solution (60 mL) of 5-(dimethylamino)-
naphthalene-1-sulfonyl chloride (dansyl chloride) (0.151 g, 0.56 mmol)
at room temperature for 40 min. The pH value of the reaction was
kept between 10 and 12 by addition of an appropriate amount of NaOH
aqueous solution (20 wt %). Then, the reaction was heated to 35 °C
with stirring for 1 h. After evaporating the solvent under a reduced
pressure, the residue was dissolved in methanol (5 mL), which was
then dropped into anhydrous diethyl ether (100 mL) under stirring. A
white precipitation was collected by suction filtration. The product was
oven at 40 °C for 24 h, a pale yellow powder (0.0070 g) was obtained.
Yield: 30%. IR (KBr pellet, cm-1): 3021, 3116, 2925, 1641, 1504,
1
1411, 1326, 781, 735. H NMR: (CDCl , ppm) δ7.597-8.235 (72H,
3
aromatic ring), δ3.700 (8H, 8-CH -CH (-CO-)-NH-), δ2.873 (16H,
2
8-NH-CH -CH -N(-CH -)-CH -), δ3.483 (16H, 8-CH -CH -
2
2
2
2
2
2
NH-CH(-CO-)-CH -), δ3.323 (8H, 4-NH-CH -CH -NH-CH-
2
2
2
), δ2.654 (32H, 16-CH -N(-CH -)-CH -CH -), δ2.366 (24H, 12-
2
2
2
2
dissolved in methanol (10 mL) and dialyzed in a dialysis bag (M
000) using methanol as a solvent at room temperature for 24 h. After
W
CH -CO-). MALDI-TOF MS (matrix: 2,5-dihydroxybenzoic acid)
2
1
Calcd for C223H220N O [M]: 3821.7. Found: 3846 (M + Na) .
+
34
28
evaporating methanol under reduced pressure and drying in a vacuum
PAMAM-Py G0 and G2-5 were synthesized in the same manner
oven at 40 °C for 24 h, a pale yellow powder (0.05 g) was obtained.
as PAMAM-Py G1. Their spectral characterization was provided in
1
Yield: 23%. H NMR: (CDCl
3
, ppm) δ6.984-8.528 (48H, aromatic
1
the Supporting Information. H NMR spectra indicated that the reaction
2
extent of amino group (-NH ) on the dendrimer surface with N-(1-
pyrenyl)maleimide was 95% for G0, 65% for G1, 95% for G2, 98%
for G3, 73% for G4, and 90% for G5.
(
24) (a) Wang, J. F.; Jia, X. R.; Zhong, H.; Luo, Y. F.; Zhao, X. S.; Cao, W.
X.; Li, M. Q. Chem. Mater. 2002, 14, 2854-2858. (b) Fluorescence
quantum yields were calculated from the integrated intensity under the
r r r
emission band A using the following equation: φ ) φ (A/A )(OD /OD)-
PAMAM dendrimers G0-5 with terminal mono- and bis-naphthyl
chromphores (PAMAM-MNp G0-5 and PAMAM-BNp G0-5) were
synthesized in a similar manner as PAMAM-Py G1 except for the
starting materials. Their spectral characterization was provided in the
2
2
(
r
n /n ), where OD is the optical density of the solution at the excitation
wavelength, and n is the refractive index. The optical density of the solution
for the calculation of quantum yields was less than 0.1 at the excitation
wavelength. The solvent 9,10-diphenylanthrene in cyclohexane was used
as reference (φ
114.)
r
) 0.90) (Eaton, D. F. Pure Appl. Chem. 1988, 60, 1107-
1
1
Supporting Information. H NMR spectra indicated that the reaction
15182 J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
extents of amino group (-NH
2
) on the dendrimer surface with N-(1-
coupling in their ground state. However, in sharp contrast, their
fluorescence spectra are quite different.
naphthyl) maleimide were 93.5%, 83%, 89%, 95%, 70%, and 83% for
PAMAM-MNp G0-5, respectively, and 90%, 92%, 90%, 86%, 86%,
and 76% for PAMAM-BNp G0-5, respectively.
For PAMAM-Py G0-5, a broad structureless fluorescence
peak was observed at 468 nm, which is well-known to be
attributable to the formation of π-π interaction between
adjacent parallel-oriented pyrene chromophores. The excimer
emission increases from G0 to G3 as shown in Figure 1,
suggesting that the higher generations favor the parallel ar-
rangement between peripheral pyrene rings. However, the
excimer fluorescence of PAMAM-Py G4 and G5 has lower
intensities than that of the third generation. This may be due to
the fact that the peripheral chromophores of the fourth and fifth
generations (G4 and G5) are more sterically crowded, which in
turn prevents the pyrene rings from parallel arrangement.
Such a fluorescence behavior is further quantified using a
widely applied fluorescence intensity ratio (IE/IM) method
Results and Discussion
Absorption and Fluorescence Spectroscopy. A. Model
Compounds. To better understand the spectroscopic properties
of the peripherally aromatic-modified dendrimers, a series of
mono- and bis-substituted model compounds, i.e., MPy, BPy,
MNp, and BNp, were synthesized by controlling the feed radios
in a Michael addition of isobutylamine with N-(1-naphthyl)-
maleimide or N-(1-pyrenyl)maleimide.
2
5
according to Winnik et al.. The intensity ratio (IE/IM) was
calculated by taking the ratio of the integrals under the
fluorescence band of 435-555 nm for the pyrene excimer (IE)
to that of 372-435 nm for the pyrene monomer (IM). The inset
of Figure 1 shows that the IE/IM value increases and reaches a
maximum at the third generation, suggesting that the parallel
arrangement is optimized at the third generation. In addition,
the IE/IM ratios were found to not depend on the dendrimer
concentration, indicating that the formation of pyrene excimer
is due to an intramolecular process of the dendrimers. These
results manifest the strong effect of dendritic architectures on
2
6a
26b
the fluorescence spectra. According to Tomalia, Turro,
2
6c
27
Goddard, and Zimmerman, the lower generations of den-
drimers are highly asymmetric and tend to exist in an open and
extended form; in contrast, the higher generations adopt a three-
dimensional globular structure with a densely packed chro-
mophore shell. Therefore, further analysis of the changes in the
IE/IM value reveals an extended-to-globular architectural transi-
tion occurring at the third generation.
The absorption and emission spectra of BPy and MPy display
the same peak shape and position as shown in Figure 1. The
24b
fluorescence quantum yield (Q) and lifetime (τ) of BPy (Q
0.120 and τ ) 72.6 ns) are about 2 times larger than those
)
of MPy (Q ) 0.0592 and τ ) 29.5 ns). This may be attributed
to a sharp increase in the molecular rigidity of BPy due to the
steric crowding of two pyrene rings.
It is very interesting to note that our results are slightly
different from those reported by Crooks and Baker for poly-
(
propylene imine) dendrimers with a peripheral pyrene chro-
For MNp and BNp, their absorption and emission spectra do
not exhibit a significant difference. The fluorescence quantum
yield and lifetime of BNp (Q ) 0.203 and τ ) 7.81 ns) are
slightly higher than those of MNp (Q ) 0.199 and τ ) 7.78
ns), suggesting that the two naphthyl rings of BNp do not lead
to a large increase in molecular rigidity as BPy. In addition, it
was found that the bis-substituted model compounds BPy and
BNp do not show excimer fluorescence, implying that two
pyrene or naphthalene rings may not form an intramolecular
parallel geometric arrangement. A conformational analysis of
the energy minimum of BPy and BNp (calculated with a
semiempirical AM1 method using a Gaussian98 program) also
reveals that the two pyrene or naphthalene rings are not parallel
but almost perpendicular to each other with twist angles of 76.1°
and 93.4°, respectively.
1
4
mophore. In their system, the excimer emission of pyrene
increases continuously from G2 to G5. The highest generation
G5 displays the strongest excimer emission. Such a difference
could be readily rationalized by considering the difference in
the dendritic backbones. The excimer formation is known to
be strongly affected by its initial excited-state placement within
the dendrimer framework according to Moore and co-workers.1
In our system, PAMAM dendrimer is relatively more rigid
because of the internal H-bonding interactions as compared with
the poly(propylene imine) in Crooks’ system. At the fourth and
fifth generations, PAMAM dendrimer may not be flexible
enough to effectively optimize the parallel arrangement of
pyrene rings.
5b
To develop a better understanding of the fluorescence
behavior of PAMAM-Py, the fluorescence decay was measured
by using a time-correlated single-photon counting technique.
B. PAMAM Dendrimers G0-5 Peripherally Modified
with Pyrenyl Chromophore (PAMAM-Py G0-5). Figures 1,
3
, and 4 show the absorption and emission spectra of the
(
25) (a) Winnik, F. M.; Regismond, S. T. A. Colloids Surf., A 1996, 118, 1-39.
(b) Winnik, F. M. Chem. ReV. 1993, 93, 587-614. (c) Capek, I. AdV.
Colloid Interface Sci. 2002, 97, 91-149.
dendrimers of the zeroth to fifth generations peripherally
modified with aromatic chromophores. For these dendrimers,
there is little peak broadening or shift in the absorption spectra
with increasing generation, suggesting that these peripheral
chromophores are well separated with very weak electron
(
26) (a) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A. Angew. Chem., Int.
Ed. 1990, 29, 138-175. (b) Caminati, G.; Turro, N. J.; Tomalia, D. A. J.
Am. Chem. Soc. 1990, 112, 8515. (c) Naylor, A. M.; Goddard, W. A. J.
Am. Chem. Soc. 1989, 111, 2339.
(27) Zeng, F. W.; Zimmerman, S. C. Chem. ReV. 1997, 97, 1681-1712.
J. AM. CHEM. SOC. VOL. 126, NO. 46, 2004 15183
9

A R T I C L E S
Wang et al.
generation increases, the torsional and rotational mobility of
the naphthyl group was hindered to cause an increase in
molecular rigidity, which thus results in an increased quantum
yield. (2) The amidoamine branches of dendritic backbone are
2
9
known to have a fluorescence quenching effect. These two
factors lead to the lowest quantum yields for PAMAM-MNp
G2, where the relatively rigid and globular structures are not
yet formed, and the fluorescence quenching of dendritic
backbone dominates. For the dendrimers with terminal bis-
naphthyl chromophores PAMPAM-BNp G0-5, their quantum
yields do not depend on the generations as shown in the inset
of Figure 3. The weak or negligible effect of dendritic
architectures may be due to the fact the peripheral adjacent
naphthyl groups are always close to each other at all generations
of PAMAM-BNp. For PAMAM-Ph G0-5, their fluorescence
is too weak to warrant a meaningful quantitative analysis.
D. PAMAM Dendrimers G0-5 Peripherally Modified
with Dansyl Chromophores (PAMAM-DNS G0-5). The
above dendrimers possess nonpolar naphthyl, pyrenyl groups
at their periphery. The emission behavior of these aromatic
chromophores is not sensitive to their surrounding polarity. In
contrast, it is well-known that, in the case of the polar dansyl
group, its emission position, shape, and intensity are sensitive
to local hydrophobicity, mobility, solvation, polarity, viscosity,
Figure 2. Fluorescence decay curves of PAMAM-Py G0-5 and mono-
and bis-substituted model compounds MPy and BPy in dichloromethane.
-
6
[
Py] ) 2.5 × 10 M. λex) 335 nm. λem) 378 nm. Inset: Average
fluorescence lifetimes 〈τ〉 of pyrenyl monomer and excimer of PAMAM-
Py G0-5 in dichloromethane at the emission wavelengths of 378 and 468
nm, respectively. λex ) 335 nm.
Figure 2 shows that the average fluorescence lifetimes 〈τ〉28 of
monomeric pyrene emission of PAMAM-Py at 378 nm increase
with increasing generation. A rapid rise in the lifetime was found
from G2 to G3 as shown in the inset of Figure 2, implying a
sharp increase in molecular rigidity from G2 to G3. This
observation further supports the assertion that the extended-to-
globular transition of dendritic architecture occurs between G2
and G3. Compared with the model compounds MPy and NPy,
the average fluorescence lifetimes and quantum yields of
PAMAM-Py G0-5 are generally lower. This could be attributed
to the quenching effect of amidoamine branches of PAMAM
dendrimers according to Pistolis and co-workers.29 However,
the average lifetimes of the pyrene excimer emission at 468
nm do not have a simple correlation with generation as shown
in the inset of Figure 2.
+
and free volume as well as H ion concentration (pH) according
7,30
to V o¨ gtle and co-workers. To further explore the relationship
between dendrimer architectures and spectroscopic behavior,
we peripherally modified the PAMAM dendrimer G0-5 with
polar dansyl chromophores and investigated their fluorescence
behavior. Figure 4 shows the emission spectra of PAMAM-
DNS G0-5. Interestingly, the fluorescence emission at a shorter
wavelength of about 413-450 nm decreases gradually, and the
emission at a longer wavelength of about 512 nm increases with
increasing generation. The second and third generations display
dual fluorescence bands at about 448 and 512 nm.
C. PAMAM Dendrimers G0-5 with Terminal Mono- and
Bis-naphthyl Chromophores (PAMAM-MNp G0-5 and
PAMAM-BNp G0-5). Figure 3 shows the absorption and
emission spectra of the dendrimers bearing peripheral naphthyl
chromophores PAMAM-MNp G0-5. There is a typical emis-
sion band of naphthyl group at 335 nm. No excimer emission
was observed for these dendrimers at all generations. The result
is consistent with that reported for polyether dendrons with
naphthyl chromophores by Fox and Stewart.1
The dansyl group is a typical twisted intramolecular charge
transfer (TICT) fluorophore according to Rettig and co-
3
1
workers. When the dansyl group is located in a nonpolar or
hydrophobic microenvironment, the dimethylamino moiety
mostly takes a coplanar conformation with the naphthyl group
at the excited state, which is termed as a locally excited (LE)
state. The maximum emission wavelength of the LE state is
about 430 nm. When the dansyl group is located in a polar or
hydrophilic microenvironment, the dimethylamino moiety mostly
takes a twisted conformation with the naphthyl group at the
excited state to form a twisted intramolecular charge transfer
5d
24
The inset of Figure 3 shows the fluorescence quantum yields
of PAMAM-MNp and PAMAM-BNp G0-5. Time-resolved
fluorescence measurements indicate that the average fluores-
(
TICT) state. The maximum emission wavelength of the TICT
2
8
cence lifetimes of PAMAM-MNp G0-5 are 5.26, 4.67, 1.19,
.15, 5.28, and 3.68 ns, respectively. PAMAM-MNp G2 exhibits
2
5c
state is about 520 nm.
5
Figure 4 shows a spectral shift and transform from about 445
nm to 512 nm in the same solvent with increasing generations,
implying that the coplanar LE state was gradually converted to
a TICT excited state from the low to the higher generations. In
the lowest quantum yield and fluorescence lifetime at all
generations. This may arise from two factors: (1) As the
(28) Average decay time 〈τ〉 was calculated from the following equation:
(
30) (a) V o¨ gtle, F.; Gestermann, S.; Kauffmann, C.; Ceroni, P.; Vicinelli, V.;
Balzani, V. J. Am. Chem Soc. 2000, 122, 10398-10404. (b) V o¨ gtle, F.;
Gestermann, S.; Kauffmann, C.; Ceroni, P.; Vicinelli, V.; de Cola, L.;
Balzani, V. J. Am. Chem. Soc. 1999, 121, 12161-12166.
The parameters of the fits were optimized by using the Marquardt-
Levenberg algorithm. Press, W. H.; Flannery, B. P.; Teukolsky, S. A.;
Vetterling, W. E. T. Numerical Recipes. The Art of Scientific Computing-
(31) (a) Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Chem. ReV. 2003, 103,
3899-4031. (b) Rettig, W.; Strehmel, B.; Schrader, S.; Seifert, H. Applied
Fluorescence in Chemistry, Biology and Medicine; Springer-Verlag: Berlin,
1999. (c) Rettig, W.; Lapouyade, R.; Lakowicz, J. R. Topic In Fluorescence
Spectroscopy. Volume 4: Probe Design and Chemical Sensing. Plenum
Press: New York, 1994; pp 109-149. (d) Maus, M.; Rettig, W.; Bonafoux,
D.; Lapouyade, R. J. Phys. Chem. A 1999, 103, 3388-3401.
(
Fortran Version); Cambridge University Press: New York, 1992. The
2
quality of the fits was estimated from the ø , the residuals, and the
autocorrelation function of the residuals.
(
29) Pistolis, G.; Malliaris, A.; Paleos, C. M.; Tsiourvas, D. Langmuir 1997,
1
3, 5870-5875.
15184 J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
Figure 3. Absorption and emission spectra of PAMAM-MNp G0-5 as well as mono- and bis-substituted model compounds MNp and BNp. The spectra
-
5
are normalized to the naphthyl chromophore concentration of 0.5 × 10 M. Inset: Fluorescence quantum yields of PAMAM-BNp and PAMAM-MNp
G0-5 in methanol, acetonitrile, and dichloromethane.
2
3
ν - ν ) (2/hc)(∆f)[(µ - µ ) /a ] + constant (1)
a
f
E
G
-
1
where νa and νf are the wavenumbers (cm ) of the absorption
and emission, respectively; µG and µE are the dipole moment
of ground state and excited state, respectively; ∆f is a solvent
parameter termed as the orientation polarizability.33
According to the Stokes’ shift (∆ν ) νa - νf) in polar and
3
3
2
3
nonpolar solvent (∆f), the value (2/hc)[(µE - µG) /a ] was
determined from the slope of the plot of ∆ν against ∆f using
eq 1. On the basis of the Onsager cavity radius (a ) 0.366 nm)
of the dansyl group as reported by Letard and Rettig,34 the
changes (∆µ ) µE - µg) in dipole moments between the first
excited singlet state and the ground state were obtained as shown
in the inset of Figure 4. There is a trend that the ∆µ increases
from G0 (4.01 D) to G3 (4.54 D) for the coplanar LE state. For
the TICT excited state of higher generations, the same trend
was observed from G2 (5.67 D) to G5 (7.16 D), which displays
a larger change in dipole moment than the coplanar LE state. It
Figure 4. Fluorescence emission spectra of PAMAM-DNS G0-5 in
-5
CH2Cl2 at the concentration 0.5 × 10 M. Inset: Changes in dipole moment
of the ground (µG) and excited states (µE) of dansyl groups at the different
generations.
-
18
should be noted that one Debye unit (1.0 D) is 1 × 10
esu
cm, and 5.67 D is comparable to the dipole moment that results
other words, the changes in dendritic architecture induce the
conversion between the two excited states. At the second and
third generations, where the extended-to-globular transition
occurs, the observation of dual fluorescence bands suggests the
coexistence of the coplanar LE and twisted TICT excited states.
The conversion from the LE to TICT state can be reasonably
explained as follows: At the higher generations, the conforma-
tion of the dansyl group tends to be slightly twisted due to the
steric crowding at the ground state. Upon photoexcitation, the
pretwisted conformation readily leads to a TICT excited-state
according to Rettig et al..31a,d
-10
from a charge separation of one unit charge (4.8 × 10
esu)
by 1.18 Å. These data are close to ∆µ ) 5.48 D for
-(dimethylamino)naphthalene-1-sulfonic acid [2-(vinyloxy-
5
35
methylamino)ethyl]amide reported by Ren and Tong and
µ ) 5.78 D for dansyl-monoaza-18-crown-6 reported by Wu
∆
36
and co-workers. From these results, it is reasonable to conclude
that the dendritic architectures at different generations have a
strong influence on the excited state of peripheral dansyl
chromophores.
Fluorescence Quenching. To gain further insights into the
effect of dendritic architectures, we investigated the fluorescence
In principle, the fluorescence behavior in various solvents
can reflect the changes in the excited state of chromophores,
which can be described quantitatively by Lippert equation as
follows,
(
33) Solvent parameters (∆f) of tetrahydrofuran, dichoromethane, N,N-dimeth-
ylformamide, methanol, and acetonitrile were calculated to be 0.2097,
0.2183, 0.2755, 0.3085, and 0.3054, respectively, according to the equation
3
2
2
2
∆
f ) (ꢀ - 1)/(2ꢀ + 1) - (n - 1)/(2n + 1), where ꢀ and n are the dielectric
constant and refractive index of solvents, respectively.
(34) Letard, J. F.; Lapouyade, R.; Rettig, W. Pure Appl. Chem. 1993, 65, 1705-
1712.
(
35) Ren, B. Y.; Gao, F.; Tong, Z.; Yan, Y. Chem. Phys. Lett. 1999, 307, 55-
(
32) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum
61.
Press: New York, 1999; p 239.
(36) Wu, Y.; Li, L.; Tong, A. Chin. Sci. Technol. Lett. 1998, 4, 196-197.
J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004 15185

A R T I C L E S
Wang et al.
Meijer,38 which leads to an effective static quenching as
illustrated in Figure 5.
Table 1. Stern-Volmer Constants (kqτ0) and Bimolecular
Quenching Constants (kq) of the Fluorescence Quenching of
PAMAM-Py G0-5 by N,N-Dimethylphenylamine and PAMAM-BNp
G0-G5 and PAMAM-MNp G0-5 by Triethylamine
Aggregation Behavior. A. Atomic Force and Transmission
Electron Microscopy of the Dendrimer Assemblies. The
peripherally aromatic modified PAMAM dendrimers with
hydrophobic shell and hydrophilic aminoamine interior are
expected to display an amphiphilic property. Therefore, the self-
assembly of these amphiphilic dendrimers was studied by means
of atomic force microscopy (AFM) and transmission electron
microscopy (TEM). Figures 6 and 7 show the AFM and TEM
images of PAMAM-Ph G0-2 assemblies in water. The spherical
aggregates or vesicles of ca. 100 nm in diameter were observed.
The aggregate size falls into the range of the typical vesicular
PAMAM-Py
G0
G1
G2
G3
G4
G5
-
1
Kqτ0/M
35.9
1.225
30.8
1.01
31.68
0.86
17.22
0.27
53.45
0.85
24.4
0.34
kq/M s-1 (109)
-
1
PAMAM-BNp
G0
G1
G2
G3
G4
G5
-
1
kqτ0/M
121
23.1
53.9
11.5
51.0
6.56
179.0
34.4
176.0
33.3
180.0
48.9
kq/M s-1 (109)
-
1
PAMAM-MNp
39
size of traditional surfactants and amphiphilic diblock copoly-
G0
G1
G2
G3
G4
4
0
mers (30-100 nm).
-
1
kqτ0/M
329
62.6
186
39.8
182
23.4
52
10.0
74
14.0
kq/M s-1 (109)
-
1
Interestingly, a large number of multiple aggregates, i.e.,
twins” and “quins” consisting of two and five individual
“
aggregates, were observed for PAMAM-Ph G1 and G2,
respectively, as shown in Figure 6b and c. The mechanism for
the formation of these remarkable multiple aggregate “twins”
and “quins” will be discussed in the following section. For
higher generations G4 and G5, we did not observe the formation
of any ordered aggregates in water.
Figure 7 compares the TEM images of PAMAM-Ph G1
aggregates with and without negative staining. It is clearly
identifiable that the “twins” and “quins” aggregates possess a
bilayer vesicular structure. The hydrophobic interphase of bilayer
appears in gray due to the high electron density of aromatic
chromophores before staining as shown in Figure 7b. After
electron staining with uranyl acetate, an exactly reversed image
was observed as shown in Figure 7a. The hydrophobic inter-
phase of bilayer was shown as relatively lighter features. The
other water-soluble portion appears in black because of the
staining by uranyl acetate. In addition, it was found that most
quenching of PAMAM-Py by N,N-dimethylphenylamine and
PAMAM-MNp and PAMAM-BNp by triethylamine. The kinet-
ics of steady-state fluorescence quenching is described by the
Stern-Volmer equation:32 I0/I ) 1+ kqτ0[Q], where I0 and τ0
are the unquenched intensity and lifetime, and [Q] is the
quencher’s concentration. The quenched intensity (I) was
corrected to avoid the inner filter effects due to the absorption
of quenchers.3 It is important to note that the correction for
the quenched intensity is very necessary when the absorption
of quenchers is close to the excitation wavelength. Some
7a
unreasonable results (e.g., kq . 101
0
M
-1 -1
s ) would be readily
The Stern-Volmer
37b,c
obtained if the data were not corrected.
plots for the fluorescence quenching were given in the Sup-
porting Information. The Stern-Volmer constant (kqτ0) and
bimolecular quenching constant (kq) were summarized in Table
1
.
“
twins” aggregates disappear after the staining as shown in
In principle, kq reflects the quenching efficiency or the
accessibility of the quenchers to the fluorophore.3 The kq values
2
Figure 7a, suggesting that the electron staining process may have
an influence on the aggregate formation.
for the zeroth generation PAMAM-MNp, PAMAM-BNp, and
PAMAM-Py are close to 1010
-1
-1
In contrast to the generally observed spherical vesicles, the
PAMAM-Py dendrimers were found to self-organize into
triangle-like vesicles in water (Figure 8). White striation could
be clearly identified from TEM images, which corresponds to
the hydrophobic interphase (lh) between bilayers with a thickness
of ca. 2.5 nm. Although the vesicles had a relatively thin wall
and varied shapes, they are very stable as evidenced by the
observation that the vesicular morphology exists over a con-
siderably long time (∼10 days). This may be attributed to the
strong π-π interaction of pyrene rings in hydrophobic part and
H-bonding interactions in hydrophilic part of the dendrimers.
Figure 9 gives a representative TEM image of PAMAM-MNp
G0 aggregates in water. The average sizes of PAMAM-MNp
and PAMAM-BNp aggregates observed by AFM and TEM are
listed in Table 3. It was found that the average diameter of these
aggregates decreases gradually from G0 to G3. The thickness
of hydrophobic interphase (lh) directly observed from TEM is
M
s , indicating a diffusion-
controlled quenching. This suggests that the dendrimers of low
generations have a more extended conformation, which is readily
accessible to the quencher. With increasing generation from G0
to G3, the kq value becomes smaller due to the steric effect.
The third generation dendrimers tends to be globular in
conformation, hence, more shielded from quenchers. The
collisional dynamic quenching occurs only if the quenchers
approach the peripheral fluorophores from a particular direction.
At the higher generations, i.e., G4 and G5, the kq value was
found to be generally greater. In addition, at higher quencher
concentrations, the I0/I ratio deviated from the linear Stern-
Volmer plot. A similar phenomenon was also observed by
Moore and Devadoss.1 This can be well understood from the
architectural effect. The globular architectures of G4 and G5
with a densely packed shell can encapsulate and bind the
quencher molecules such as a “dendritic box” as described by
5e
(
38) Jansen, J. F. G. A.; de Brabander-van den Berg, E. M. M.; Meijer, E. W.
(
37) (a) To avoid the inner filter effects due to the absorption of the incident
Science 1994, 266, 1226-1229.
light and the emitted light, the fluorescence intensity was corrected as
(39) (a) Marrink, S. J.; Mark, A. E. J. Am. Chem. Soc. 2003, 125, 15233-
15242. (b) Myers, D. Surfaces, Interfaces, and Colloids: Principles and
Applications, 2nd ed.; John Wiley & Sons: New York, 1999.
(40) (a) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (b) Zhang,
L. F.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777-1779. (c) Zhang, L.
F.; Eisenberg, A. Science 1995, 268, 1728-1731.
[
(ODex+ODem)/2]
follow: I ) Iobsd × 10
. Iobsd is the observed fluorescence
intensity. ODex and ODem are the optical densities at excitation and emission
wavelength, respectively. (b) Phelan, J. C.; Sung, C. S. P. Macromolecules
1
1
997, 30, 6845-6851. (c) Phelan, J. C.; Sung, C. S. P. Macromolecules
997, 30, 6837-6844.
15186 J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
Figure 5. Schematic illustration for more extended architecture at low generations (e.g., G1) with an open and accessible structure by quenchers (left) and
the globular architecture at higher generations (e.g., G4) with a densely packed shell (right). The closed globular structure could encapsulate and bind some
quenchers in its interior but sterically hinder outer quenchers from entering into its interior.
in a range of 2.3-3.4 nm. The bilayer thickness in a range of
.6-6.4 nm was thus obtained from the hydrophobic thickness
attached vesicles leads to the structures from “twins I” to “twins
II” as shown in Figures 6c, 10, and 11.
3
(
(
lh) plus 2 times the maximum extended length of dendrimers
ld). The data are close to the thickness of natural lipid
In contrast to the vesicles from traditional amphiphilic
compounds, these dendrimer-based vesicles have several distinct
features. (1) They possess a very thin wall (hydrophobic
interphase of 2.3-3.4 nm) as compared with the vesicles from
diblock copolymers (10-20 nm). (2) The driving force for the
self-organization may be the π-π interaction of aromatic
moieties and H-bonding interaction of dendrimer branches. (3)
The driving force of self-organization along with further
entanglement of the dendrimer branches leads to the formation
of a large number of multivesicle “twins” and “quins” with a
significant degree of internal stability.
For the fourth and fifth generations of these dendrimers,
however, no ordered aggregates or vesicles were observed
(Figure S-12 in the Supporting Information). This is expected
due to the fact that the dendrimers G4 and G5 possess a globular
architecture with a compact surface. The sterical crowding and
rigid dendritic architecture are not flexible enough to form
ordered assemblies. Recently, Meijer and Stevelmans22 also
reported the absence of the ordered superstructures at higher
generations (e.g., G4 or higher).
membranes (∼5 nm) and the bilayer thickness (4.8-5.4 nm)
of vesicles from poly(propylene imine) dendrimer bearing long
2
2a
hydrophobic soft chains reported by Meijer and Schenning.
The bilayer thickness is much lower than those for the vesicles
4
0
made from amphiphilic block copolymers (10-20 nm). So
far, there have been many vesicle-forming species reported such
41a
as hydrophilic modified fullerenes,
cationic and anionic
surfactants, and various amphiphilic block copolymers.41c To
the best of our knowledge, this is the first observation of the
bilayer vesicle formation from the hydrophilic dendrimers
bearing short hydrophobic rigid chromophores at their periphery.
The formation of bilayer vesicles can be attributed to the
amphiphilic structure of dendrimers, i.e., hydrophilic dendritic
branches and hydrophobic aromatic periphery. At the low
generations (G0-3), the open and extended architectures
facilitate the changes in shapes of the dendrimers. The hydro-
philic branches of dendrimers tumble out to occupy more room
in water. To minimize the energy in water, the hydrophobic
aromatic moieties of dendrimers shrink and pack together in
parallel with the aid of π-π interaction to form the hydrophobic
interphase between bilayers as illustrated in Figure 10.
41b
B. Critical Aggregation Concentration, Aggregation Num-
ber, and Critical Packing Parameter. To further elucidate the
mechanism for the aggregate formation, we have measured some
key aggregation parameters. The critical aggregation concentra-
tion (CAC) of these dendrimer aggregates was determined by
following the change in fluorescence intensity or the ratio (IE/
The bilayer structure of vesicles was clearly confirmed by
their TEM images with and without negative staining (Figures
7, 8, and 11). The outer and inner layers of the vesicles appear
2
5,32
in black from TEM images with negative staining, indicating
that the portion consists of the hydrophilic dendritic branches.
The bilayer interphase appears in relative gray from the TEM
images without negative staining, indicating that the interphase
consists of hydrophobic aromatic moieties with high electron
density.
Because the flexible hydrophilic dendritic branches are located
in the outer layer, these vesicles are very adhesive. They are
readily attached by H-bonding interaction and the entanglement
of the branches. Therefore, a large number of aggregate “twins”
consisting of two vesicles, even multivesicle “quins” consisting
of five individuals, were observed. Further fusion of two
IM) with increasing dendrimer concentration.
The original
plots are shown in Figure S-8 in the Supporting Information.
The CAC values obtained are summarized in Table 2.
-5
-7
All the CAC values are very low (10 -10 M) as expected
for these amphiphilic dendrimers. The CAC value appears to
decrease in the order of PAMAM-MNp > PAMAM-BNp >
PAMAM-Py. PAMAM-Py self-organizes into ordered ag-
gregates more readily than PAMAM-BNp and PAMAM-MNp.
This indicates that pyrene rings or larger aromatic chromophores
favor the self-organization process due to the stronger π-π
interaction. In addition, the CAC value decreases gradually from
G0 to G3, suggesting that the aggregates are favorably formed
in the order of G3 > G2 > G1 > G0. Within the range of
G0-3, the higher generations favor aggregation because of the
increase in aromatic rings and dendrimer branches.
(
41) (a) Zhou, S. Q.; Burger, C.; Chu, B.; Sawamura, M.; Nagahama, N.;
Toganoh, M.; Hackler, U. E.; Isobe, H.; Nakamura, E. Science 2001, 291,
1
944-1947. (b) Wang, X. H.; Goh, S. H.; Lu, Z. H.; Lee, S. Y.; Wu, C.
Macromolecules 1999, 32, 2786-2788. (c) Jung, H. T.; Coldren, B.;
Zasadzinski, J. A.; Iampietro, D. J.; Kaler, E. W. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 1353-1357.
Currently, there are several techniques to obtain the informa-
tion on the aggregate characteristics, such as a light scattering
J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004 15187

A R T I C L E S
Wang et al.
Figure 6. AFM images (topography and phase) of the PAMAM-Ph G0 (a), G1 (b) and G2 (c) aggregates deposited on the mica surface, displaying the
multiple aggregate “twins” and “quins” consisting of two and five individual aggregates.
technique as reported by Chu, Zhou, and Wu41 and fluorescence
quenching technique as reported by Strauss and Thomas. We
attempted to utilize the above techniques to determine the
aggregation numbers but have not been successful. The ag-
gregation numbers determined by fluorescence techniques based
on a probe/quencher combination are generally accepted for
surfactant-like spherical micelles. The basic principle of this
technique is the Poisson statistical quenching when the probe
and quencher molecules are partitioned into micellar micro-
4
3
4
4
(
42) To obtain reliable data, the thickness of hydrophobic interphase was
measured from TEM images without electron staining. The reason for this
is it was found that the thickness observed from TEM images with negative
staining is slightly larger than the actual thickness. This might be due to
the negative uranyl acetate staining. In TEM images with negative staining,
only the accessible area of the electron stainer was observed in black, which
makes the relatively lighter area (apparent hydrophobic interphase) look
slightly larger.
(43) (a) Chu, D.; Thomas, J. K. Macromolecules 1987, 20, 2133-2138. (b)
Hsu, J.; Strauss, U. P. J. Phys. Chem. 1987, 91, 6238-6241. (c) Zdanowicz,
V.; Strauss, U. P. Macromolecules 1993, 26, 4770-4773.
15188 J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
Figure 7. TEM images of the PAMAM-Ph G1 aggregates in water with
negative staining (a) where the hydrophilic part was stained in black and
without electron staining (b) that displays a reversed image. Concentra-
tion: 0.5 mg/mL.
domains. However, the exact measurement for some complex
aggregates, such as cylindrical micells, microemulsions, vesicles,
and bilayers, has been debated.45 Although the laser light
scattering technique can provide some useful information on
aggregation behavior, it contains many hypotheses, which often
leads to unreliable results.4
1b
In the present study, we used the geometric relation model
proposed and developed by Tanford46 and Israelachvili to
quantify the shape and size of aggregates. In brief, the geometric
characteristics of an individual amphiphilic dendrimer should
be strongly correlated to the shape and size of its aggregates.
There are three critical geometric characteristics of the individual
dendrimer as shown in Figure 10: (1) the optimal area occupied
by the hydrophilic branches of dendrimers (a0); (2) the volume
of the individual dendrimer (V); and (3) the maximum extended
length (ld), which is comparable to the radius of the dendrimer
47
3
molecule. Based on the dendrimer density 1.232 g/cm as
Figure 8. TEM images of triangle-like aggregates of PAMAM-Py G0
without electron staining (a) and PAMAM-Py G1 (b), G2 (c), and G3 (d)
with negative staining in water. Concentration: 0.5 mg/mL.
reported by Tomalia,48 the volume of the individual dendrimer
(V) can be determined from its molecular weight (M). The
average sizes of the vesicles and the average thickness of the
hydrophobic interphase between bilayers were determined from
50 transmission images (about 900 aggregates). From the
average size and thickness, the aggregation number of inner
and outer layers can be determined by dividing the overall
occupied volume by the volume of the individual dendrimer.
The a0 value is subsequently obtained from the overall spherical
area divided by the aggregation number.
(
44) (a) Hansson, P.; Schneider, S.; Lindman, B. J. Phys. Chem. B 2002, 106,
9
777-9793. (b) Bales, B. L.; Zana, R. J. Phys. Chem. B 2002, 106, 1926-
1939. (c) Vasilescu, M.; Caragheorgheopol, A.; Caldararu, H. AdV. Colloid
Interface Sci. 2001, 89, 169-194. (d) Hansson, P.; Jonsson, B.; Strom, C.;
Soderman, O. J. Phys. Chem. B 2000, 104, 3496-3506. (e) Rodenhiser,
A. P.; Kwak, J. C. T. J. Phys. Chem. B 1999. 103, 2970-2972.
45) (a) Kramer, M. C.; Steger, J. R.; Hu, Y. X.; McCormick, C. L.
Macromolecules 1996, 29, 1992-1997. (b) Hu, Y. X.; Armentrout, R. S.;
McCormick, C. L. Macromolecules 1997, 30, 3538-3546.
(
(
(
(
46) Tanford, C. The Hydrophobic Effect. Formation of Micelles and Biological
From the above data, the critical packing parameter (Pc) can
Membranes, 2nd ed.; Wiley: New York, 1980.
47,39b
47) . Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic
Press: San Diego, CA, 1991.
be calculated using the equation: Pc ) V/a0lc.
The Pc value
is a key parameter to predicting the shape and size of
48) . Topp, A.; Bauer, B. J.; Tomalia, D. A.; Amis, E. J. Macromolecules 1999,
3
9
3
2, 7232-7237.
aggregates that will produce a minimum in free energy for a
J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004 15189

A R T I C L E S
Wang et al.
cence resonance energy transfer (FRET) between pyrene and
naphthalene chromophores to obtain further characterization on
these dendrimer-based self-assemblies. FRET is known to
32
depend on the following three factors: (1) the extent of spectral
overlap of acceptor absorption and donor emission, (2) the
quantum yields of the donor, and (3) the distance between the
donor and acceptor. FRET is a long-range and through-space
interaction, which is independent of the intervening medium
between donor and acceptor, such as solvents, chemical bonds,
50-52
and polymer backbones.
The distance dependence of FRET
has been employed as a “spectroscopic ruler” for a variety of
applications, such as distance measurement between donor and
5
1a,b
51c,d
51e,f
acceptor,
macromolecular associations,
protein folding,
conformational changes,
5
1g
membrane fusion, and lipid
Figure 9. Representative TEM image of PAMAM-MNp G0 aggregates in
water. Concentration: 0.5 mg/mL.
51h
exchange. The photophysical phenomenon of FRET between
donor and acceptor covalently attached to a polymer has been
termed “photo harvesting” by Webber and the “antenna effect”
by Guillet. The naphthalene/pyrene energy transfer pair has
been employed to study the compatibility, thermo-reversible
52
given amphiphilic dendritic structure. According to Israelach-
vili,47 bilayer vesicular structures can be generated only if 0.5
e Pc e 1. If Pc < 0.33, spherical micelles can be formed. If
5
3
54a
phase transition, and steric hindrance of polymers by Morawetz,
0
.33e Pc e 0.5, one can predict the formation of cylindrical or
5
4b
52b
Winnik, and Webber. In our previous work, we attempted
to employ FRET to measure the bilayer thickness of vesicles
made from amphiphilic copolymers. But, it is unsuccessful. The
reason for this is that the bilayer thickness of vesicles from
amphiphilic copolymer is generally in the range of 100-200
Å, which exceeds the effective range measured by FRET (20-
disk-shaped micelles. If Pc > 1.0, reversed or inverted micelles
can be formed. These packing parameters provide the detailed
aggregate characteristics of dendrimers as listed in Table 3. The
Pc values for PAMAM-MNp G0-3 were found to be in the
range of 0.5-1.0 and tend to decrease with increasing genera-
tion, suggesting that the aggregates possess bilayer vesicular
structures. The theoretical prediction is in agreement with the
actual observation from TEM. The same trend was observed
for PAMAM-BNp aggregates.
9
0 Å).
In the present study, we have designed a control experiment
in an effort to determine the bilayer thickness of dendrimer-
based vesicles by changing the ratio of PAMAM-Py to
PAMAM-MNp aggregates in the mixture for FRET measure-
For PAMAM-MNp aggregates, their average aggregation
4
3
numbers (〈N〉) decrease from 7.9 × 10 for G0 to 4.0 × 10 for
-
4
4
ment. Thus, 9.0 mL of PAMAM-Py aggregates (1.5 × 10
G3, which is close to the aggregation number (∼1.2 × 10 ) of
M) was mixed with 1.0 mL of PAMAM-MNp aggregates (1.5
bilayer vesicles made from C60 fullerene bearing hydrophilic
-
4
groups reported by Zhou and Chu.41a The aggregation numbers
× 10 M). At the excitation wavelength of 290 nm where the
radiation is mostly absorbed by the naphthalene moiety, the
fluorescence of the mixed aggregates was observed mostly from
the pyrene chromophore with a relatively weak residual
fluorescence of the naphthalene chromophore as shown in the
inset (A and B) of Figure 12. This indicates that FRET occurs
from the naphthalene to pyrene chromophore.
of these dendrimer- and C60-based vesicles are considerably
larger than those of the vesicles derived from traditional ionic
〈N〉 )50-100)4
factants as well as diblock copolymers (〈N〉 ) 50-1000).
9a,b
49c,d
(
and nonionic (〈N〉 ) 100-1000)
sur-
4
9e-g
This is understandable because of their common features:
instead of the flexible hydrophobic tails of traditional am-
phiphilic surfactants and diblock copolymers, both dendrimer-
and C60-based amphiphilic compounds possess rigid and bulky
hydrophobic moieties with a dominant intrinsic geometric
constraint. At the same generation, PAMAM-MNp aggregates
show the larger aggregation numbers and Pc value than
PAMAM-BNp aggregates. This is attributed to the relative size
of hydrophobic and hydrophilic portions of individual den-
drimer. The hydrophobic headgroups of PAMAM-BNp are
bulky and more crowded as compared to that of PAMAM-MNp.
For PAMAM-Py aggregates, these packing parameters are
difficult to be assessed reliably because they are nonspherical.
Dendrimer Exchanges and Fluorescence Resonance En-
ergy Transfer. As an in-depth study, we utilized the fluores-
Figure 12 shows the time-resolved fluorescence spectra of
-
4
the mixed aggregates of PAMAM-MNp G1 (1.5 × 10 M,
.0 mL) and PAMAM-Py G1 (1.5 × 10 M, 9.0 mL). At the
excitation wavelength of 295 nm and emission wavelength of
45 nm, the fluorescence decay was fitted by a single-
-
4
1
3
(50) Calzaferri, G.; Huber, S.; Maas, H.; Minkowski, C. Angew Chem., Int. Ed.
2003, 42, 3732-3758.
(51) (a) Brogan, A. P.; Widger, W. R.; Kohn, H. J. Org. Chem. 2003, 68, 5575-
5587. (b) Cha, A.; Snyder, G. E.; Selvin, P. R.; Bezanilla, F. Nature 1999,
402, 809-813. (c) Lipman, E. A.; Schuler, B.; Bakajin, O.; Eaton, W. A.
Science 2003, 301, 1233-1235. (d) Schuler, B.; Lipman, E. A.; Eaton, W.
A. Nature 2002, 419, 743-747. (e) Rice, S.; Lin, A. W.; Safer, D.; Hart,
C. L.; Naber, N.; Carragher, B. O.; Cain, S. M.; Pechatnikova, E.; Wilson-
Kubalek, E. M.; Whittaker, M.; Pate, E.; Cooke, R.; Taylor, E. W.; Milligan,
R. A.; Vale, R. D. Nature 1999, 402, 778-784. (f) Remy, I.; Wilson, I.
A.; Michnick, S. W. Science 1999, 283, 990-993. (g) Varma, R.; Mayor,
S. Nature 1998, 394, 798-801.(h) Mochizuki, N.; Yamashita, S.; Kuroka-
wa, K.; Ohba, Y.; Nagai, T.; Miyawaki, A.; Matsuda, M. Nature 2001,
411, 1065-1068.
(
49) (a) Benrraou, M.; Bales, B. L.; Zana, R. J. Phys. Chem. B 2003, 107,
1
3432-13440. (b) Srinivasan, V.; Blankschtein, D. Langmuir 2003, 19,
(52) (a) Webber, S. E. Chem. ReV. 1990, 90, 1469-1482. (b) Fox, S. L.; Chan,
J. C.; Kiserow, D. J.; Ramireddy, C.; Munk, P.; Webber, S. E. AdV. Chem.
Ser. 1996, 248, 141-162.
(53) Guillet, J. E.; Rendall, W. A. Macromolecules 1986, 19, 224-230.
(54) (a) Morawetz, H. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 1725-
1735. (b) Mizusaki, M.; Morishima, Y.; Winnik, F. M. Macromolecules
1999, 32, 4317-4326. (c) Zhao, C. L.; Wang, Y.; Hruska, Z.; Winnik, M.
A. Macromolecules 1990, 23, 4082-4087. (d) Winnik, F. M. Macromol-
ecules 1989, 22, 734-742, 55.
9
932-9945. (c) Li, Y.; McMillan, C. A.; Bloor, D. M.; Penfold, J.; Warr,
J.; Holzwarth, J. F.; Wyn-Jones, E. Langmuir 2000, 16, 7999-8004. (d)
Kamenka, N.; Chevalier, Y.; Zana, R. Langmuir 1995, 11, 3351-3355.
(
e) Alexandridis, P.; Hatton, T. A. Colloid Surf., A 1995, 96, 1-46. (f)
Yusa, S.; Sakakibara, A.; Yamamoto, T.; Morishima, Y. Macromolecules
002, 35, 10182-10188. (g) Lysenko, E. A.; Bronich, T. K.; Slonkina, E.
V.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Macromolecules 2002,
5, 6344-6350.
2
3
15190 J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
Figure 10. Schematic illustration of the vesicular formation and “twins I” and “twins II” structures for PAMAM-MNp G1. Two spherical vesicles were
attached by the entanglement and H-bonding interaction of dendritic branches to form “twins I”. Further fusion leads to the formation of the “twins II”
structure. Hydrophobic aromatic chromophores are shown in blue, and the hydrophilic branches are in black.
Table 3. Spherical Aggregate Characteristics of PAMAM-MNp
G0-3 and PAMAM-BNp G0-3 in Water
PAMAM-MNp
G0
G1
G2
G3
-
1
M/g mol
1409
90.3
3214
6807
14 044
40.8
18.9
1.65
2.7
a
<
R>outer /nm
82.7
4.33
1.01
3.0
58.6
9.18
1.30
3.4
3
b
V/nm (per dendrimer) 1.90
c
ld/nm (per dendrimer)
0.768
2.3
d
〈
〈
〈
lh〉 /nm
N〉outere
N〉inner
4
4
3
3
3
4.1 × 10 2.0 × 10 5.9 × 10 2.2 × 10
4
4
3
3.8 × 10 1.8 × 10 5.0 × 10 1.8 × 10
2
f
a0/nm (per dendrimer) 2.5
4.3
0.997
7.1
0.995
12
0.955
g
Pc
0.989
Figure 11. Representative TEM images of “twins II” structures for
PAMAM-BNp G1 (a) and G2 (b) aggregates and TEM image without
negative staining of “twins I” structure for the mixed aggregates of
PAMAM-BNp
-
4
G0
G1
G2
G3
PAMAM-MNp G1 (1.5 × 10 M, 1.0 mL) and PAMAM-Py G1 (1.5 ×
-
4
1
(
0
M, 9.0 mL) (c) displaying the thickness of hydrophobic interphase
-1
M/g mol
2303
68.9
5000
10379
48.3
14.0
1.50
3.3
21188
40.6
28.6
1.90
3.2
4
2
3.1 ( 0.1 nm).
a
<
R>outer /nm
57.5
6.74
1.17
3.2
3
b
V/nm (per dendrimer) 3.10
Table 2. Critical Aggregation Concentration (CAC) of PAMAM-Py
G0-3 and PAMAM-MNp G0-3 in Water
c
ld/nm (per dendrimer)
0.905
2.5
d
〈
lh〉 /nm
e
4
3
3
3
3
PAMAM-Py
〈N〉outer
1.7 × 10 7.1 × 10 3.0 × 10 1.3 × 10
4 3 3
〈
N〉inner
1.5 × 10 6.0 × 10 2.5 × 10 0.9 × 10
G0
G1
G2
G3
2
f
a0/nm (per dendrimer) 3.5
5.8
9.4
16
9.70 × 10-
6
3.67 × 10-6
2.10 × 10-7
1.52 × 10-7
g
CAC/M
Pc
0.979
0.993
0.993
0.941
PAMAM-MNp
a
Average radius 〈R〉 of aggregates directly observed from AFM and
TEM. b Volume of the individual dendrimer (V). Maxmum extended length
ld) of dendrimers, which is comparable to the radius of dendrimer molecule.
c
G0
G1
G2
G3
(
-
5
1.25 × 10-5 0.98 × 10-5 1.24 × 10
-6
CAC/M 1.70 × 10
d
Average thickness 〈lh〉 of hydrophobic interphase between bilayer directly
-
6 a
-7 a
observed from TEM. e Average aggregation numbers of outer and inner
(
7.43 × 10
)
(7.41 × 10
)
f
monolayer (〈N〉outer and 〈N〉inner). Optimal area occupied by the hydrophilic
a
branches of dendrimers (a ). g Critical packing parameter (P ) V/a l ).
CAC value for PAMAM-BNp aggregates.
0
c
0 d
there is an exchange of dendrimer molecules between ag-
gregates. Therefore, these aggregates are not isolated from each
other.
exponential curve. The fitted lifetime is 8.87 ns assigned to the
emission lifetime of naphthalene chromophore. With increasing
emission wavelength, the decay curve becomes broader. The
fluorescence decay was fitted by a dual exponential curve. An
increasing amount of relative long lifetime (31.4 ns) emission
was observed, which is assigned to the emission lifetime of
pyrene chromophores. The time-resolved spectra confirm that
the nonradiative energy transfer occurs from the naphthalene
to pyrene chromophore in the mixed aggregates, suggesting that
It is well-known that the diffusion of the individual molecule
of the outer monolayer is much easier than that of the inner
39a
monolayer as described by Marrink and Mark and that the
individual molecules located in the outer layer almost do not
3
9b
exchange with ones in the inner layer of vesicles. Therefore,
the exchanges of individual dendrimer due to its diffusion mostly
occurred in the outer monolayer of vesicular aggregates. For
J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004 15191

A R T I C L E S
Wang et al.
-
4
-4
Figure 12. Time-resolved fluorescence decay of the mixed aggregates of PAMAM-MNp G1 (1.5 × 10 M) and PAMAM-Py G1 (1.5 × 10 M) in a 1:9
volume ratio in H2O. The excitation wavelength was 290.0 nm, and the emission wavelength was varied from 300.0 to 460.0 nm in 3.0 nm steps. Inset:
Steady-state three-dimensional fluorescence (emission-excitation-intensity) spectra of PAMAM-MNp G1 aggregate (1.5 × 10 M) before (A) and after (B)
-
4
-
4
mixing PAMAM-Py G1 aggregates (1.5 × 10 M) in a 1:9 volume ratio.
-
4
to be 3.521 × 10 M-1 cm (nm) from the absorption
spectrum of PAMAM-MNp G0 aggregates and the emission
spectrum of PAMAM-Py G0 aggregates, after correction for
the nonradiative self-quenching of adjacent naphthyl or pyrenyl
chromophores within the same monolayer. The spectral overlap
integral calculated for other dendrimer generations is essentially
the same. The R0 value was subsequently calculated to be 20.8
( 0.2 Å. The transfer efficiency (E) was determined by
calculating the quantum yields of naphthyl chromophores of
PAMAM-MNp aggregates before (QD) and after (QAD) mixing
with PAMAM-Py aggregates according to the following equa-
14
-1
4
the mixed aggregates of PAMAM-MNp (1.5 × 10 M)/
-4
PAMAM-Py (1.5 × 10 ) in a 1:9 volume ratio, such exchanges
should result in about 10% of mixed bilayer vesicles, where
the inner monolayer mostly consists of naphthalene chro-
mophores, while the outer monolayer mostly consists of pyrene
chromophores as illustrated in Figure 13. When a naphthalene
chromophore is excited at 290 nm, the fluorescence emission
from pyrene was indeed observed at 378-400 nm due to FRET.
Other aggregates (about 90%) mostly consist of pyrene chro-
mophores with a few naphthalene chromophores. They almost
do not absorb and are not excited at 290 nm. FRET from pyrene
to naphthalene is negligible.
3
2
tion.
The FRET from naphthyl chromophores of the inner layer
to the pyrenyl chromophores of the outer layer can be thus
E ) 1 - QAD/QD
(3)
49
employed as a spectroscopic ruler to measure the hydrophobic
interphase thickness of the mixed vesicles. When the efficiency
of FRET is 50%, the distance between acceptor and donor is
called the F o¨ rster distance.32 On the bases of the absorption
and emission spectra of model compounds MNp and MPy,
F o¨ rster distance (R0) was calculated by using the following
equation:32
-
4
For the mixed aggregates of PAMAM-MNp G0 (1.5 × 10
-4
M) and PAMAM-Py G0 (1.5 × 10 M) in a 1: 9 volume ratio,
the E value is calculated to be 0.46. For PAMAM-MNp G1/
PAMAM-Py G1 under the same mixing condition, the E value
is calculated to be 0.11. For PAMAM-MNp G2/PAMAM-Py
G2, the E value is 0.07. For PAMAM-MNp G3/PAMAM-Py
G3, the E value is 0.21. Based on the R0 and E values, the
average distance (r) between naphthyl and pyrenyl chro-
mophores was determined by using the following equation:
∞
6
0
2
5
4
4
R ) [9000(ln 10)k Q ]/128π Nn [
∫
F (λ) ꢀ (λ) λ dλ]/
D
D
A
0
∞
6
2 -4
[
∫
F (λ) dλ] ) 0.211 [k n Q J(λ)] (2)
D
D
6
6
6
0
E ) R /(R + r )
(4)
0
0
where QD is the fluorescence quantum yields of the energy donor
naphthalene, n is the refractive index of the solvent, J(λ) is the
spectral overlap integral, ꢀA is the molar extinction coefficient
of the acceptor pyrene, and FD(λ) is the fluorescence spectrum
of the donor naphthalene on a wavelength (λ) scale. The R0
value forthe MNp-MPy pair was calculated to be 26.0 Å, which
is in the range 20-60 Å for a typical donor-acceptor pair. This
The average thickness of hydrophobic interphase between
bilayer is equal to the average distance (r), which was calculated
to be 2.04 nm for the PAMAM-MNp G0/PAMAM-Py G0
mixed aggregates, 2.95 nm for PAMAM-MNp G1/PAMAM-
Py G1, 3.20 nm for PAMAM-MNp G2/PAMAM-Py G2, and
2
.59 nm for PAMAM-MNp G3/PAMAM-Py G3, respectively
as shown in Figure 13.
4
5
value is close to 26.6 Å reported by McCormick and
To further confirm the fluorescence results, the mixed
aggregates were examined with TEM to measure the interphase
thickness directly. As shown in Figure 11c, the average thickness
of hydrophobic interphase measured from TEM is 3.1 ( 0.1
5
4c,d
55
Winnik
and Ringsdorf for a naphthalene/pyrene pair. For
the dendrimers, the spectral overlap integral J(λ) was calculated
(
55) Ringsdorf, H.; Sackmann, E.; Simon, J.; Winnik, F. M. Biochim. Biophys.
42
Acta 1993, 1153, 335-344.
nm, which is in excellent agreement with 2.95 nm from FRET.
15192 J. AM. CHEM. SOC.
9
VOL. 126, NO. 46, 2004

Peripherally Modified Poly(amidoamine) Dendrimers
A R T I C L E S
-
4
-4
Figure 13. Schematic illustration of dendrimer exchanges for the mixed aggregates of PAMAM-MNp G1 (1.5× 10 M) and PAMAM-Py G1 (1.5× 10
M) in water in a 1:9 volume ratio. In the mixed bilayer structures (∼10%), the outer monolayer mostly consists of pyrenyl chromophores shown in red, and
the inner monolayer mostly consists of naphthyl chromophores shown in blue. Fluorescence resonance energy transfer (FRET) occurs from the inner to the
outer monolayer upon photoexcitation at 290 nm. Other aggregates (∼90%) mostly consist of pyrenyl chromophores, where there is almost no emission
observed at the excitation wavelength 290 nm.
The slightly lower value might be due to the fact the thickness
measured from FRET is a center-to-center distance of aromatic
chromophores of hydrophobic interphase, while the thickness
observed from TEM is an edge-to-edge distance as illustrated
in Figure 13.
units, and further entanglement of dendrimer branches. For these
dendrimers with peripheral pyrenyl chromophores, irregular
triangle-like vesicles were observed in water. In contrast to the
vesicles from traditional surfactants and amphiphilic block
copolymers, the dendrimer-based vesicles have several distinct
features: (1) The vesicles possess a thin wall (e.g., hydrophobic
interphase of ca. 2.3-3.4 nm) as compared to that from
amphiphilic block copolymers (10-20 nm). (2) The vesicles
are very adhesive due to the dendrimer branches located in the
outer layer. A large number of interesting “twins” and “quins”
vesicular aggregates consisting of two and five vesicles,
respectively, were observed for the first time. The average
diameter and aggregation number of the vesicles were found to
decrease with increasing generation. The critical aggregation
concentration value shows that the vesicle formation is favored
in the order of G3 > G2 > G1 > G0. However, higher
generations, i.e., G4 and G5, do not form ordered aggregates.
Based on the fluorescence resonance energy transfer between
Conclusions
PAMAM Dendrimers G0-5 were peripherally modified with
naphthyl, pyrenyl, and dansyl chromophores. Their fluorescence
behavior was found to depend strongly on the dendritic
architecture and correlate with the transition of dendritic
architecture from an extended structure at the lower generations
to a three-dimensional globular shape at the higher generations
(e.g., G4 or higher). AFM and TEM images show that these
amphiphilic dendrimers at the low generations (G0-3) can self-
organize into bilayer vesicles in water. The self-organization is
likely induced by the π-π interaction of the peripheral aromatic
chromophores, the H-bonding interaction of interior amidoamine
J. AM. CHEM. SOC.
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A R T I C L E S
Wang et al.
naphthyl and pyrenyl chromophores in a mixed vesicle system,
an intervesicular exchange of dendrimer molecules was revealed,
and the hydrophobic interphase thickness of the mixed vesicles
was determined to be 2.04-3.20 nm, which agrees well with
the thickness observed with TEM.
PAMAM-Ph G2-5, PAMAM-DNS G0, G2-5, PAMAM-Py
G0, 2-5, PAMAM-MNp G0-5, PAMAM-BNp G0-5; Stern-
Volmer plots for the fluorescence quenching of PAMAM-BNp
by triethylamine, PAMAM-MNp by triethylamine, and PAMAM-
Py by N,N-dimethylphenylamine; critical aggregation concentra-
tion of PAMAM-Py G3, PAMAM-BNp G3, and PAMAM-MNp
G3 determined by fluorescence methods; reaction dynamic
curves of the peripheral modification of PAMAM G2 and G3;
fluorescence spectra of PAMAM-MNp G2-4 with different
reaction extents; AFM images of the PAMAM-Py G4-5 and
PAMAM-BNp G5 aggregates deposited on the mica surface.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. We are most grateful to the National
Natural Science Foundation of China (NSFC Grant No.
2
0340420002 and No. 20374002) for the financial support.
Partial support from the Nanotechnology Institute of South-
eastern Pennsylvania is also acknowledged.
Supporting Information Available: Materials, measurements,
spectral characterization of model compounds MNp, BNp, MPy,
BPy and peripherally modified PAMAM dendrimers, i.e.,
JA048219R
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VOL. 126, NO. 46, 2004