Non-ionic dendronized multiamphiphilic polymers as nanocarriers for biomedical applications

Article abstract of DOI:10.1002/smll.201201253

A new class of non-ionic dendronized multiamphiphilic polymers is prepared from a biodegradable (AB)_n-type diblock polymer synthesized from 2-azido-1,3-propanediol (azido glycerol) and polyethylene glycol (PEG)-600 diethylester using Novozym-43

Full text of DOI:10.1002/smll.201201253

full papers
Polymers
Non-ionic Dendronized Multiamphiphilic Polymers as
Nanocarriers for Biomedical Applications
Shilpi Gupta, Boris Schade, Sumit Kumar, Christoph Böttcher, Sunil K. Sharma,*
and Rainer Haag*
A new class of non-ionic dendronized multiamphiphilic polymers is prepared from a
biodegradable (AB)n-type diblock polymer synthesized from 2-azido-1,3-propanediol
(azido glycerol) and polyethylene glycol (PEG)-600 diethylester using Novozym-435
(Candida antarctica lipase) as a biocatalyst, following a well-established biocatalytic
route. These polymers are functionalized with dendritic polyglycerols (G1 and G2)
and octadecyl chains in different functionalization levels via click chemistry to
generate dendronized multiamphiphilic polymers. Surface tension measurements
and dynamic light scattering studies reveal that all of the multiamphiphilic polymers
spontaneously self-assemble in aqueous solution. Cryogenic transmission electron
microscopy further proves the formation of multiamphiphiles towards monodisperse
spherical micelles of about 7–9 nm in diameter. The evidence from UV–vis and
fluorescence spectroscopy suggests the effective solubilization of hydrophobic
guests like pyrene and 1-anilinonaphthalene-8-sulfonic acid within the hydrophobic
core of the micelles. These results demonstrate the potential of these dendronized
multiamphiphilic polymers for the development of prospective drug delivery systems
for the solubilization of poorly water soluble drugs.
multicomponent polyplexes use specific water-soluble poly-
1. Introduction
mers, either as a bioactive component itself or as an inert
functional part of a multifaceted construct for improved drug,
protein, or gene delivery.[1–5] Current approaches towards
solubilizing active components involve non-ionic polymeric
materials such as Cremophor, Tween, and Pluronics, which
have been extensively used to formulate hydrophobic guest
Several supramolecular architectures on the nanoscale level
have become important candidates for potential appli-
cations in nanomedicine, particularly for drug, dye, and
gene delivery.[1–5] Polymeric drugs, polymer–drug conju-
gates, polymer–protein conjugates, polymeric micelles, and
Dr. S. Gupta, Dr. S. Kumar, Prof. R. Haag
Institut für Chemie und Biochemie
Freie Universität Berlin
Takustraße 3, 14195 Berlin, Germany
E-mail: haag@chemie.fu-berlin.de
Dr. B. Schade, Dr. C. Böttcher
Forschungszentrum für Elektronenmikroskopie
Institut für Chemie und Biochemie
Freie Universität Berlin Fabeckstraße 36a
14195 Berlin, Germany
Dr. S. Gupta, Prof. S. K. Sharma
Department of Chemistry
University of Delhi
Dr. S. Kumar
Department of Chemistry
Deenbandhu Chhotu Ram University of Science & Technology
Murthal (Sonipat), Haryana-131039, India
Delhi-110 007, India
E-mail: sk.sharma90@gmail.com
DOI: 10.1002/smll.201201253
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Non-ionic Dendronized Multiamphiphilic Polymers as Nanocarriers
molecules.[2b,6] Biocompatiblity and biodegradablity of these benefits of PEG-type polymers, which have long clearance
nanocarriers are especially important for the clinical applica- times, high stability and solubility, no immunogenicity, anti-
tion of therapeutic compounds.
genicity, or toxicity,[18,19] better monodispersity than classical
The research on the encapsulation of hydrophobic mole- monomolecular amphiphiles,[17e] and enhanced stability and
cules has indicated the need to improve drug-delivery proc- biocompatibility due to polyglycerol dendrons.[2a] In contrast
esses with the right transporter architectures with more to polysoaps where monomolecular amphiphiles are lined
precise biodistribution and a better pharmacokinetic profile up within polymers,[20] the layout of such a polymer should
for targeting specific diseases (especially tumors, via the EPR allow an independent aggregation of headgroups and tails
(enhanced permeation and retention) effect).[7] Thus, there is due to the flexibility of the polyethylene oxide (PEO) chains
a quest to develop larger sized, well-defined, and biocompat- and thus lead to stable supramolecular nanocarriers.
ible molecular objects as prospective drug-delivery systems.
Therefore, a new class of dendronized multiamphiphilic
Out of various conceptual extensions for the development of polymers was synthesized, starting with an (AB)n-type
such molecular objects, one approach uses polymers as mul- polymer from two biocompatible synthons and then grafting
tifunctional, polydisperse cores to which dendrons are con- to it hydrophilic polyglycerol dendrons as headgroups as well
nected via pendant functional groups at every repeat unit as hydrophobic alkyl chains as tails. A modular biocatalytic
and are termed ‘dendronized polymers’ or ‘denpols.’[8,9]
synthetic approach was used to polymerize azido glycerol
Dendronized polymers were originally termed ‘rod- and PEG-600 diethyl ester with Novozym-435. Click cou-
shaped dendrimers,’ and made their first appearance in 1987 pling with different generation dendrons and hydrophobic
in a patent filed by Tomalia et al.[10] From their work on den- alkyl chains gave well-defined multiamphiphiles that self-
drimer synthesis, this group set out to synthesize larger highly assembled at low critical aggregation concentrations. All the
functionalized structures. Hawker and Fréchet created hybrid new dendronized multiamphiphilic polymers were tested as
architectures of dendrimers and linear polymers by attaching potential nanocarriers for drug-delivery applications using
Fréchet-type dendrons to a styrene derivative and copolym- the hydrophobic fluorescent dye pyrene. Structural param-
erizing it with styrene.[11] The predominant role of geometry eters influencing the solubilization properties of hydro-
in the self-assembly of polymers equipped with ‘taper-shaped’ phobic guests were investigated by varying the hydrophobic/
side chains was recognized by Percec et al.[12] Extensive work hydrophilic balance and the interaction with photochromic
on synthesis and characterization of dendronized polymers guests was studied using the fluorescent probe pyrene and
was carried out by the Schlüter group who held the world 1-anilinonaphthalene-8-sulfonic acid (ANS).
record in shape persistant dendronized polymers.[9,13] They
synthesized rod-like polymers with a conjugated backbone
and recognized the significance of dendron decoration for
the backbone conformation.[13c]
2. Results and Discussion
We have designed a biocatalytic approach for the synthesis of
biodegradable (AB)n-type diblock polymer (3) from 2-azido-
1,3-propanediol (azido glycerol, 1) (Scheme 1) and PEG-
600 diethylester (2) using Novozym-435 (Candida antarctica
lipase) as biocatalyst (Scheme 2). A modular approach was
developed for synthesis of structurally varied (dendronized)
polymers (10a–f) by making use of highly efficient click
coupling reactions to attach different generation dendrons
and alkyl chains (Scheme 2). This study is focused on the
supramolecular self-assembly of these dendronized multiam-
phiphilic polymers, in particular, the effect of dendron loading
onto linear PEG-based polymers and the ability to solubilize
hydrophobic guest molecules.
Dendronized polymers are now being used both as new
materials in already established applications, e.g., as organic
materials for optoelectronic applications, as well as in
intriguing new concepts which may only be accessible with
this remarkable class of molecule.[8,14] They have helped to
bring different fields of research together.[9,15] Amphiphilic
dendronized polymers with nanoscopic dimensions are
among the most interesting target structures,[8] but have not
yet been much explored for drug-delivery applications.
Our interest was to extend the multiamphiphilic PEGs[6]
with structurally defined glycerol dendrons to synthesize bio-
compatible dendronized multiamphiphilic polymers. We have
recently developed novel biocatalytic approaches for the
synthesis of PEG/glycerol/polyglycerol-based polymers and
explored their potential as delivery vehicles.[16] We have also
developed a variety of amphiphilic architectures based on
dendritic polyglycerol (dPG) that self-assemble in aqueous
2.1. Synthesis and Characterization
solution under physiological conditions into stable aggre- 2-Azido-1,3-propanediol (azido glycerol) (4) was synthesized
gates with a nanoscopic size range for the delivery of active from commercially available glycerol in four steps (Scheme 1).
agents.[6,17] Recently, it became evident that the introduction Glycerol was first converted to 2-hydroxypropane-1,3-diyl dia-
of polyglycerol dendrons in monomolecular amphiphiles has cetate (1) using vinyl acetate as the acylating reagent, by fol-
a remarkable stabilizing effect, resulting in structurally very lowing the established procedure.[21] The secondary hydroxyl
defined supramolecular architectures.[17e]
group of 2-hydroxypropane-1,3-diyl diacetate (1) was then
We assumed that polyglycerol dendrons would also mesylated (3) and subsequently an azidation reaction was per-
improve the overall structural stability of polymer based formed to obtain 2-azidopropane-1,3-diyl diacetate (3). The
supramolecular architectures if covalently linked to a linear compound 3 was deacetylated using K2CO3 in anhydrous eth-
polymer backbone. This would combine the biomedical anol to obtain the monomer 2-azido-1,3-propanediol (4).
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Scheme 1. Multi-gram synthesis of 2-azido-1,3-propanediol (4).
The structure of compound 4 was confirmed on the basis 13C NMR spectrum disappeared completely. The peaks for
of its spectral data (IR, 1H and 13C NMR, distortionless methine carbon showed characteristic downfield shifts from
enhancement by polarization transfer (DEPT)-135, and high- δ 58.38 to δ 66.58 on deacetylation; methine and the meth-
1
resolution mass spectra (HRMS). In its H NMR spectrum, ylene carbons were further corroborated with the DEPT-135
the methine and the methylene protons recorded upfield NMR spectrum.
shifts from δ 3.89–3.83 and δ 4.22–4.09 in the precursor 2-azi-
Lipases have been employed for their versatile catalytic
dopropane-1,3-diyl diacetate to δ 3.50–3.47 and δ 3.70–3.55, efficiency for esterification/transesterification reactions and
respectively. The characteristic signals for the acetyl moiety at biocatalytic polymerization reactions.[22,23] Drawing upon our
1
δ 2.08 in H NMR spectrum and at δ 20.56 and 170.33 in the previous work on the catalyzed condensation of PEG-600
Scheme 2. Synthesis of polymer 6 from 2-azido-1,3-propanediol (4) and PEG-600 diethylester (5), followed by the click chemistry approach to
synthesize grafted polymers (10a–f) from alkyne-functionalized [G1.0] (8) or [G2.0] (9) dendrons and octadecyl propargyl ether (7). Inset represents
the click coupling of polymer 6 and self-assembly of the dendronized multiamphiphilic polymers (10c–f) in aqueous media.
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Non-ionic Dendronized Multiamphiphilic Polymers as Nanocarriers
dimethylester and glycerol under solventless conditions,[16a]
Novozym-435 was further employed for the polymerization
of 2-azido-1,3-propanediol (4) and PEG-600 diethylester
(5), which was synthesized from its corresponding diacid by
a standard esterification protocol[16a] to afford the polymer
poly[(2-azidopropan)-1,3-dioxy-poly(oxyethylene-600)oyl)]
(6) at 83% yield (Scheme 2). Use of the diester 5 instead
of the diacid not only enhances the degree of polymeriza-
tion but also introduces an ethyl group that could be used
for end-group analysis to determine the average molecular
1
weight of the polymer by H-NMR. Polymer 6 was unam-
biguously characterized from its IR, 1H, 13C, DEPT-135, and
2D-NMR spectral data. In its 1H NMR spectrum, the C-1
and C-3 protons recorded downfield shifts to δ 4.37–4.11
with respect to the monomer 2-azido-1,3-propanediol (4)
(δ 3.70–3.55). This downfield shift is attributed to the trans-
esterification reaction during polymerization, and occurs
due to the attached primary hydroxyl groups. Protons at
C-2 exhibit a downfield shift to δ 3.81–3.43 (compared to δ
3.50–3.47 in 4). Characteristic signals for the PEG chain pro-
tons and ethyl end group of PEG-600 diethylester (5) were
observed at δ 3.81–3.43 and δ 1.28–1.24, respectively. Assign-
ments of signals were confirmed by 1H-13C heteronuclear
Figure 1. Exemplary graph of surface tension (γ) versus log concentration
of the dendronized multiamphiphile 10f in aqueous solution at 25 °C.
The functionalized polymers were unambiguously iden-
multiple quantum coherence (HMQC) correlation spectrum, tified on the basis of their spectral data (c.f. SI) as for den-
and DEPT-135 NMR spectrum analysis (see the Supporting dronized multiamphiphilic polymer 10f. In the 1H NMR
Information (SI)). From the DEPT-135 NMR spectrum the spectrum of dendronized multiamphiphilic polymer 10f,
end-group carbons of azido glycerol (1) resonating at δ 63.67, the C-1, C-3, and C-2 protons recorded a downfield shift
61.72 (C-1 and C-3), and δ 61.48 (C-2) can be distinguished and resonated at δ 4.71–4.40 and δ 5.14–5.09 with respect
from the main chain carbons, which resonate at δ 63.21 (C-1 to the precursor polymer, (δ 4.37–4.11: C-1 and C-3 protons
and C-3) and 58.45 (C-2). The presence of azide groups can and δ 3.94–3.88: C-2 protons). This significant downfield
1
further be confirmed from a signal at 2131.4 cm−1 in the IR shift in the H NMR spectra revealed that the azide func-
spectrum. The integration values for the ethyl ester protons tional group of polymer 6 was involved in click coupling
1
were correlated to the peaks for other protons in H-NMR with the alkyne functionalized side chains. Assignments of
spectrum and the degree of polymerization was observed the signals at δ 4.29–4.14 for the α and α’ protons of the
to be approximately 9. The corresponding molecular weight PEG main chain, and methoxy protons of the side chains in
1
1
was calculated to be 6294 g mol−1 , while the number-average the H NMR spectrum were confirmed by an H-1H COSY
molecular weight of 6708 g mol−1 was obtained by gel per- spectrum (SI).
meation chromatography (GPC) in tetrahydrofuran (THF)
(1 mL min−1 ). The polydispersity index (PDI) was found to
2.2. Supramolecular Self-Assembly in Aqueous Media
be 1.40. The elemental data analysis was also observed to be
in accordance (for monomeric unit (C29H53N3O16)n calcu-
lated: C, 49.78; H, 7.63; N, 6.01, observed: C, 47.73; H, 7.32;
N, 5.33).
The supramolecular self-assembly of the synthesized non-
ionic dendronized polymers 10a–f in aqueous media was
investigated using surface tension measurements, dynamic
light scattering (DLS), and cryogenic transmission electron
microscopy (cryo-TEM). Subsequently, the solubilization
capacities of the aggregates were evaluated using pyrene
as a hydrophobic fluorescent guest and the interactions
with the guest molecules were investigated by ANS binding
experiments.
Octadecyl-propargyl ether (7)[24] and alkyne function-
alized [G1.0] and [G2.0] generation polyglycerol dendrons
(8 and 9) were synthesized, following well established
procedures.[17b,25] The azide functionalized polymer 6 was
conjugated with either alkyne functionalized [G1.0] dendrons
(8) or octadecyl-propargyl ether (7) in dimethylformamide
(DMF) using copper (I) bromide as a catalyst in a one-pot
reaction (Scheme 2) to generate the grafted polymers 10a
and 10b. The series of dendronized multiamphiphilic poly-
2.2.1. Surface Tension Measurements
mers 10c–f was synthesized by varying the ratio of the side Surface tension measurements were carried out in a pendant
chains, using mixtures of desired proportions of both the drop apparatus. The data were plotted as logarithmic func-
hydrophobic (7) as well as the hydrophilic (8 or 9) alkynes. tions of the surfactant concentrations. The surface tension
Progress of the reaction was monitored by the decreasing (γ) of the aqueous solutions was effectively reduced as the
intensity of the azide signal in the IR spectrum. After stirring polymer concentration increased. A clear break in the curve
for 24 h at room temperature, the product was dialyzed to marks the critical aggregation concentration (CAC; Figure 1),
remove the unreacted starting material.
for which the values are listed in Table 1.
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T a b l e 1 . Critical aggregation concentration (CAC) of polymers 10a–f in
aqueous solution by surface tension measurements at 25 °C.
Polymer
Composition of R [%]
Alkyl chain [G1.0]-PG [G2.0]-PG
CAC
(10−5 M)
–
(mg L−1
)
–
50
–
–
–
–
10a
10b
10c
10d
10e
10f
50
50
70
50
70
2.00
2.94
2.50
2.41
2.08
153
262
224
247
203
50
30
-
–
–
50
30
-
Figure 2. Cryo-TEM images of alkyl grafted [G2.0]-PG dendronized
polymers 10e (left) and 10f (right) in water. The polymers aggregate
into monodisperse spherical micelles, which form densely packed
domains. FTs of such domains (insets) provide circular diffraction
patterns corresponding to micelles mean distances of 8.4 and 7.8 nm,
respectively. Comparing all five polymers (10b–f), the ring diameter
of 10e (inset left) is blurred to a much higher extent, indicating that
packing is not so accurate in this case. This is brought about by a less
defined hydrophilic seam of low contrast, probably introduced by the
large number of space-consuming [G2.0] dendrons within the micelles.
Scale bar is 50 nm.
As was expected from its overall hydrophilic nature,
dendronized polymer 10a completely dissolved in water and
thus did not show any CAC. Exclusively alkyl-functionalized
polymer 10b had a CAC value of 2.00 × 10−5 M (153 mg L−1 )
and the dendronized multiamphiphilic polymers which differ
in percentage of octadecyl chains and in the dendron gener-
ations, either [G1.0] (10c and 10d) or [G2.0] (10e and 10f),
have CAC values of 2.94 × 10−5 M (262 mg L−1 ) and 2.50 ×
10−5 M (224 mg L−1 ), and 2.41 × 10−5 M (247 mg L−1 ) and
2.08 × 10−5 M (203 mg L−1 ), respectively. Thus, the CAC of the
dendronized multiamphiphiles is lowered both by a larger spherical micelles as exemplified in Figure 2. It was observed
proportion of hydrophobic side chains (10d < 10c and 10f < that most of the micelles tended to accumulate into large,
10e) as well as a higher generation of the dendrons (10e < 10c densely packed domains, irrespective of compound and prepa-
and 10f < 10d). Similar correlations were found for mono- ration method. Isolated particles were monitored only very
meric non-ionic dendritic amphiphiles by Haag et al.[17e] and rarely. Such domains were analyzed by Fourier transform (FT),
for amphiphilic block copolymers composed of styrene and which gave diffraction rings (insets in Figure 2) indicative of
sodium acrylate by Eisenberg and co-workers.[26] Poly(N- the repetitive packing distance of the micelles. The values can
isopropylacrylamide-block-dendronized methacrylate) syn- be used for a quite accurate determination of the mean diam-
thesized by Schlüter et al. showed much lower CAC values, eter of the micelles as summarized in Table 2. Although the
however, ranging from 1.2–6.9 mg L−1 .[27]
diameter value only varies in a narrow margin of 8.1 0.5 nm
for the alkylated polymer (10b) and 8.4, 7.5, 8.4, and 7.8 nm
( 0.8 nm) for the four dendronized multiamphiphilic polymers
(10c–f), it is quite evident that the polymers with the higher
alkyl fractions (10d and 10f) have smaller diameters. Errors are
estimated from the width of the diffraction rings.
A closer look at the FTs of all samples (cf. SI) unveils the
interdependence of size distribution and the content of den-
drons. Thus the diffraction rings from the alkylated polymer
(10b) are quite sharp, and clearly point to only minor devi-
ations of the micelles mean diameter, while those of the
2.2.2. Cryogenic Transmission Electron Microscopy and Dynamic
Light Scattering
Aggregation behavior of the polymers was studied by cryo-
TEM and DLS at a general concentration of 10.0 g L−1 , which
is well above the CAC of the polymers. The cryo-TEM meas-
urements confirmed that exculsively dendronized polymer 10a,
completely dissolves in water without any sign of aggregation
as was already anticipated from the absence of a CAC. All
other multiamphiphilic polymers (10b–f) formed monodisperse
T a b l e 2 . PDI and particle size of the micelles 10b–f above the CAC in aqueous solution as measured by DLS (by intensity) and cryo-TEM at 25 °C.
These values are compared with those from geometrical calculations. The hydrophobic volume of the micelles was calculated to be 113.1 nm3 for
all five polymers.
Polymer
Diameters [nm]
Calculated Volumes [nm3]
NAgg
DLS
12.5
15.3
14.0
19.3
17.2
PDI
TEM
8.1
8.4
7.5
8.4
7.8
Calc.
8.4
8.8
8.2
9.2
8.4
Hydrophilic
196.2
Total
309.3
354.1
287.1
401.5
309.4
–
0.543
0.603
0.535
0.502
0.427
28.1
28.1
22.1
28.1
22.1
10b
10c
10d
10e
10f
[G1.0]
[G1.0]
[G2.0]
[G2.0]
241.0
174.0
288.4
196.3
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Non-ionic Dendronized Multiamphiphilic Polymers as Nanocarriers
dendronized multiamphiphiles (10c–f) are blurred to some hydrated functional groups like PEO are not shown. DLS, on
extent, indicating that packing is not so accurate in these the other hand, measures statistical averages of particle sizes,
cases.This is brought about by a less defined hydrophilic seam which, in contrast, not only includes all parts (atoms) of the
of low contrast due to PEG chains, which are barely visible in aggregates along with their hydration shell, but is also influ-
the images but might cause varying inter-assembly distances. enced by volume-independent properties of the aggregates
Within the dendronized multiamphiphiles, the diffraction like shape and mutual interactions. Therefore, hydrodynamic
rings from 10e (50% alkyl, 50% [G2.0]) are broadened the radii often exaggerate proper particle sizes.
most, probably because of the large number (∼127, cf. Experi-
A relevant comparsion can be made by the dendronized
mental Section, Geometrical Calculations of Micellar Dimen- polymers reported by Chen et al. They studied Fréchet-type
sions) of the more space-consuming [G2.0] dendrons, which benzyl ether dendrons with a carboxyl group (G2, G3) at the
are also not constrained in their conformation.
apex site attached to poly(4-vinylpyridine) (PVP), forming
From our earlier experiments with monomolecular hydrogen-bonded dendronized polymers. These polymers
amphiphiles[17e] we expected a significant contribution of the showed unique self-assembly behavior, forming aggregates
PG dendrons to the contrast of images for the dendronized with average diameters from 100 to 400 nm. They observed
multiamphiphilic polymers 10c–f. Anyhow, the observed that the size of the vesicles decreased and the thickness of
micelles are very similar to the micelles of the solely alkyl the vascular membrane increased with the increase in the
grafted polymer and thus seem to lack this additional high- molar ratio of the dendrons to PVP.[28]
contrast corona. This indicates less organization of the PG
dendrons on the micellar surfaces compared to the mono-
2.2.3. Structural Model of the Micelles
meric analogues, where intermolecular interactions led to
Geometrical calculations of the micelles dimensions (cf. SI)
render overall diameters of 8.4 nm (10b), 8.8 and 9.2 nm
(10c and 10e) and 8.2 and 8.4 nm (10d and 10f), and point
to approximately 127 (10c and 10e) or 60 (10d and 10f) PG
groups (whether [G1.0] or [G2.0]), respectively, within their
hydrophilic shells. As can be seen from Table 2, the calcu-
lated micellar diameters commensurate well with the average
values obtained from the Fourier transforms of the cryo-
TEM images, within the limits of error (0.5–0.8 nm). These
calculations indicate that, from the geometrical point of view,
only minor differences in the diameters can be expected for
all five aggregating polymers. Graphical models representing
the principle differences of the molecular arrangement in the
micellar systems are presented in Figure 3.
If the micelles are pushed together to form 2D ordered
arrays, the FT should yield the inter-assembly distance (center
to center), which is indicative of the overall diameter of the
micelles in the ideal case. Blurring of the diffraction rings indi-
cates variations in the particle distance, which might be due
to variations in the head group corona geometry and packing
density. Actually, the surface should be crimped with the den-
drons and the overall spherical shape might be distorted by
the mutual compression of the micelles. Therefore, it is not
surprising that the observed mean diameters (by cryo-TEM)
are slightly smaller than the calculated ones, while the hydro-
dynamic diameters should, in contrast, be slightly larger. The
presented DLS data, however, clearly exceed these theoretical
structurally defined micelles.[17e]
Cryo-TEM measurements were complemented by DLS
measurements. The exclusively alkyl grafted polymer 10b
provided hydrodynamic diameters of 12.5 nm, which is in
keeping with the value obtained from the cryo-TEM measure-
ments (8.1 nm) considering the poor visibility of the hydrated
shell. Furthermore, the DLS data display considerably higher
hydrodynamic diameters of 15.3 and 14.0 nm for the [G1.0]
multiamphiphiles 10c and 10d and 19.3 and 17.2 nm for the
[G2.0] multiamphiphiles 10e and 10f. The observed DLS data
indicates a general trend that while the more alkyl-grafted
polymers form smaller micelles, higher generation dendron
[G2.0] grafted polymers form larger ones (cf. Table 2).
Furthermore, the DLS graphs (by intensity) of all polymers
(10b–f) display components with hydrodynamic diameters
larger than 100 nm, which might be agglomerated aggregates
or free (not aggregated) polymer strands. However, the abso-
lute numbers of such particles must be in the ppm range, as
can be inferred from the absence of corresponding bands in
the volume and number distributions (cf. SI).
It should be emphasised here that the differences between
cryo-TEM and DLS data do not necessarily arise from dif-
ferent particles, since both methods display completely dif-
ferent phenomena. TEM pictures depict those parts of
individual aggregates where electron density deviates from
the surrounding medium (usually water), while strongly
Figure 3. The five graphical models represent the motifs in the spatial micellar organization of the alkyl grafted multiamphiphilic polymer (10b)
and dendronized multiamphiphilic polymers (10c–f). The micelles’ size and surface patterns alter according to the grafting ratios and the specific
generation of the linked dendrons.
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Figure 4. A l l fi ve multiamphiphilic polymers form spherical micelles, in
which the PEO spacers (top, gray) form loops to allow for a convenient
aggregation of the hydrophobic alkyl chains. Spreading of one polymer
strand across two micelles via one of those 4 nm long PEO chains (black)
leads to intermicellar chaining.
values. This points either to an extensive hydration shell, or
(depending on the generation of the hydrophilic headgroups)
to attractive interactions between micelles, which might enable
the formation of bigger clusters of micelles. Such attractive
forces can also be concluded from the ordered domains fre-
quently observed on the cryo-TEM micrographs, although the
individual number of interacting micelles remains shrouded
in the extensive 2D arrangement, which might be also intro-
duced in the sample preparation step.
Discrepancies in the aggregation behavior of the five multi-
amphiphilic polymers, depending on the type of linked den-
drons and the grafting proportions at the polymeric back-bone
(Figure 3), became apparent from the DLS measurements.
Their nature and origin, however, can only be estimated from a
detailedcomparisonofDLSandcryo-TEMdata.Thedifferences
as well as the observed clustering in cryo-TEM point to attrac-
tive interactions between the micelles, which may be induced
(1) by attractions of the [G1.0] or [G2.0] dendrons, or (2) from
intermicellar chaining due to the intercalation of the polymer
strands, probably due to the nearly 4 nm-long water-soluble
PEO spacers (Figure 4). Such intermicellar chainings are fre-
quently observed with ABA- or ABC-type copolymers in dif-
ferent media, if the B-block is solvophilic.[29–31]
Figure5. a)UVabsorptionspectra(λmax=329nm)ofpyreneencapsulated
by polymers 10a–f in aqueous solution (C = 5 mg mL−1 ); b) histogram
representing the guest solubilization capacities (npyrene/npolymer) for the
dendronized multiamphiphilic polymers 10c–f.
Dendronized polymer 10a, which lacks any hydrophobic
alkyl chains, did not incorporate the hydrophobic guest pyrene,
while polymers 10b–f have considerable guest solubilization
properties. Thus, within this group of polymers the presence of
an internal hydrophobic core is the essential structural feature
for the encapsulation of hydrophobic guest molecules.Figure 5a
shows UV spectra of pyrene in 5 mg mL−1 aqueous solutions
of different multiamphiphiles. The absorption intensity at λmax
(329 nm) is directly correlated to their guest solubilization
capacities (defined as moles of guest molecule per mole of
amphiphile). In the row of the three multiamphiphiles with the
same percentage of hydrophobic content (50% alkyl), [G1.0]
dendronized multiamphiphile 10c has the lowest solubilization
capacity (1 mol pyrene per mol of polymer), followed by non-
dendronized multiamphiphile 10b (1.4 mol pyrene per mol of
polymer) and the [G2.0] dendronized multiamphiphile 10e
(2.1 mol pyrene per mol of polymer) (Table 3). In general,
multiamphiphiles with [G2.0] dendrons (10e, 10f) provide better
guest solubilization capacities (2.1, 2.3 mol pyrene per mol of
2.2.4. Encapsulation Studies
The polymeric multiamphiphiles were evaluated for their guest
solubilization properties using pyrene as a hydrophobic guest
molecule. To establish the polymer structure with guest solu-
bilization property, the quantity of encapsulated and, hence,
solubilized pyrene in the different multiamphiphiles was
evaluated. Our focus has been to understand the effect of dif-
ferent hydrophobic (core) and hydrophilic (shell) content on
the dendronized multiamphiphilic polymers on its host–guest
properties and to evaluate the influence of the hydrophobic/
hydrophilic ratio of the multiamphiphiles on the solubilization
capacity and solubilization efficiency for lipophilic guests.
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Non-ionic Dendronized Multiamphiphilic Polymers as Nanocarriers
T a b l e 3 . Guest solubilization capacities and efficiencies of the poly-
mers 10a–f (conc. of 5 g L−1 ) for solubilizing hydrophobic guest pyrene.
the third (384 nm), and the first (373 nm) vibrational peak
in the emission spectrum).[34] The values of I3/I1 range from
0.555 in water to 1.666 in aliphatic hydrocarbon solvents.[34]
The maximum values obtained for the dendronized multiam-
phiphilic polymers 10c–f were 0.790, 0.792, 0.798, and 0.802
respectively, at a polymer concentration of 1.0 mg mL−1 , sug-
gesting that pyrene molecules are likely to be located in a
hydrophobic pocket. A value of 0.618, was reached for the
dendronized polymer 10a, and 0.832 for the non-dendronized
multiamphiphilic polymer 10b, which indicates a significantly
higher overall polar environment of pyrene in polymer 10a
and a significantly higher nonpolar environment of pyrene
in polymer 10b, respectively, compared to the dendronized
multiamphiphilic polymers 10c–f. These results can be well
understood from the structural composition of the polymers,
as the dendronized multiamphiphilic polymer 10a lacks a
hydrophobic pocket. Thus the I3/I1 value corresponds to an
environment of high polarity (most approximates to ethylene
glycol).[33] Polymers 10b–f, on the other hand, have a hydro-
phobic pocket composed of alkyl chains to solubilize pyrene
which leads to a change in its microenvironment.
Polymer
Solubilization capacity
Solubilization efficiency
[nguest/npolymer
]
[mg g−1
]
–
–
10a
10b
10c
10d
10e
10f
1.4
1.0
1.9
2.1
2.3
35.5
20.9
67.1
42.4
74.8
polymer, respectively) compared to those with [G1.0] dendrons
(10c, 10d: 1.0, 1.9 mol pyrene per mol of polymer; Figure 5b).
Thus, the generation of the dendrons plays a significant role for
the guest solubilization properties of these multiamphiphiles:
[G2.0] dendrons stabilize the micelles, as indicated by the
higher solubilization capacities, while [G1.0] dendrons seem to
destabilize them, if compared to polymer 10b.
The guest solubilization efficiency (milligrams of guest per
gram of amphiphile) increases,however,with an increased per-
centage of hydrophobic content (Table 3). The dendronized
multiamphiphiles 10c and 10e (50% alkyl grafted) encapsu-
2.3. Binding Studies with 1-Anilinonaphthalene-8-sulfonic Acid
late less pyrene (20.9 mg and 42.4 mg, respectively) than 10d The encapsulation experiments with pyrene suggest that
(67.1 mg) and 10f (74.8 mg) (70% alkyl grafted), respectively. these new multiamphiphiles can be employed for encapsula-
Altogether, our new dendronized multiamphiphilic polymers tion of active agents/drugs and may find potential biomedical
show solubilization efficiencies of up to 7.5 wt% (10f), which application as drug carriers. However, for drug delivery, it is
is considerably higher if compared with monomolecular essential to know the polymer’s binding characteristics and
non-ionic dendritic amphiphiles (up to 2 wt%),[17e] and thus, association with guest molecules. Quantitative analysis of
highlights the versatility of the dendronized multiamphiphilic the polymer molecule binding capacity and affinity can be
polymeric architectures for prospective drug delivery appli- assessed by encapsulation of small fluorescent dyes.[35] Since
cations over corresponding monomolecular amphiphiles.
all relevant multiamphiphiles 10b–f show the similar micel-
The solubilization site of pyrene in the dendronized multi- lization behaviors, the dendronized multiamphiphile 10f with
amphiphilic polymers was also studied based on the well- the most apparent guest solubilization properties was chosen
known sensitivity of pyrene fluorescence on the polarity of its for the ANS binding studies.
local environment.[32,33] Evidence for the presence of hydro-
The fluorescence of 1-anilinonaphthalene-8-sulfonic
phobic environment of pyrene is obtained from the higher acid (ANS) is sensitive to changes in the microenviron-
values of polarity index (I3/I1, the ratio of the intensities of ment,[36] which allows the evaluation of host–guest binding
Figure 6. a) ANS fluorescence intensity upon addition of polymer 10f (λmax = 477 nm) to ANS solution. CANS = 5 μM; λexc = 360 nm; b) ANS
fluorescence intensity upon addition of ANS (λmax = 477 nm) to polymer 10f solution. C10f = 20 μM; λexc = 360 nm; c) the determination of Kb and
n by Scatchard’s plot for binding of ANS to polymer 10f.
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full papers
interactions by spectrofluorometric methods. Due to dynamic to the higher ordered structure of larger dendrons, [G2.0]-
quenching by water molecules, ANS shows only low fluores- PG dendron functionalized multiamphiphiles show better
cence in the range between 400 and 600 nm with an emission guest solubilization efficiencies than [G1.0]-PG dendronized
maximum (λmax) at 525 nm in aqueous media. The fluores- multiamphiphiles and non-dendronized multiamphiphiles.
cence is greatly enhanced and is accompanied by a blue-shift Additional studies with ANS revealed unimolecular binding
of the band, as the solvents polarity decreases. Upon addition characteristics within the hydrophobic domain of the micelles
of polymer 10f the ANS fluorescence was significantly that indicates the guest molecules are predominantly located
increased (Figure 6a) and gradually led to a blue shift of λmax in the internal hydrophobic cores of the aggregates. Allto-
to about 477 nm. Since the polymer solution did not show any gether, this new class of dendronized multiamphiphilic poly-
bands in this range, these changes arose due to the binding of mers demonstrates the formation of notable uniform micelles
ANS and suggest that the dye was placed in a hydrophobic with higher guest solubilization capacities and efficiencies
environment, well shielded from the surrounding water mol- compared to monomolecular amphiphiles, added biocompat-
ecules. By further increasing the polymer concentration the ibility due to the use of PEG and PG dendrons, and lower
fluorescence began to show signs of maximizing as more ANS exchange rates due to covalently linked multiamphiphilic
was bound. From the resulting hyperbolic curve (Figure 6a), architecture, which all ought to enhance the potential use
the value of Fsp was calculated (cf. Experimental Section). as nanocarriers for biomedical applications. Further work is
The fluorescence intensity (F) was measured in the second underway to study the cellular uptake mechanism of these
fluorometric titration, where the system was reversed and new multiamphiphiles, fine-tune the structure to achieve
increasing concentrations of ANS were added to a constant more stable architectures, and obtain controlled drug release
concentration of the multiamphiphile (Figure 6b). From these profiles.
values the concentrations of bound ANS (Cbound ANS) and
free ANS (CfreeANS) were calculated and the Scatchard’s plot ration of tailor-made nanocarriers adapted to specific needs
(C10h/Cbound against 1/Cfree
in Figure 6c) was drawn. and targeted to specific tissues. Following this concept,
We believe that this new concept will facilitate the prepa-
ANS
ANS
The linear regression of the plot (Figure 6c) indicates that multi-functional nanocarriers may in the future be easily
ANS penetrates inside the micelles, where only one type of constructed by picking up the required side chains from a
binding center (or hydrophobic pocket) is located. Values for ‘construction kit’ of suited functional groups and clicking
Kb ∼ 1.66 × 105 m−1 and n ∼ 0.34 per molecule of the polymer them at a polymeric back-bone of a convenient length.
10f were calculated.
4. Experimental Section
3. Conclusion
All the chemicals and solvents used were of analytical grade and
A new class of non-ionic dendronized multiamphiphilic poly- purchased from Sigma-Aldrich GmbH (Munich, Germany) and
mers and their self-assembly to micellar nanocarriers has Fisher Scientific GmbH (Schwerte, Germany). Novozym-435 was
been presented. The polymers are constructed from biocom- purchased from Julich Chiral Solutions GmbH (Jülich, Germany).
patible PEG chains and PG dendrons to form a hydrophilic Analytical TLCs were performed on precoated Merck silica-gel
outer shell and alkyl chains moulding for a hydrophobic 60 F254 plates; silica gel (100–200 mesh) was used for column
interior. The polymers backbone was synthesized from chromatography. Milli-Q water (Millipore GmbH, Schwalbach/Ts.,
linear PEG and 2-azido-1,3-propanediol using a biocatalytic Germany, resistivity ∼18 ΜΩ.cm, pH = 5.6 0.2) was used in all
pathway. The versatility of this basic structure originates from experiments and for preparation of all samples. Dialysis was per-
its azido groups which facilitate an easy functionalization by formed using benzoylated dialysis tubing (MW cutoff 2000 Da)
click grafting with appropriate alkynes (alkyl groups or den- from Sigma-Aldrich, changing the solvent three times over a period
1
drons). This opens the way to a new class of multiamphiphilic of 24 h. H and 13C NMR spectra were recorded on a Bruker DRX
surfactants, where the hydrophobic/hydrophilic balance as 400, and a Bruker AMX 500 MHz spectrometers (Bruker GmbH,
well as functionalities may be easily readjusted as required. Karlsruhe, Germany). The spectra were calibrated by using the sol-
The flexibility of the PEG block allows for an independent vent peaks. The chemical shift values are on δ scale and the coup-
aggregation of hydrophilic and hydrophobic groups.
ling constant values (J) are in Hz. IR spectra of neat samples were
As a proof of concept, the polymeric backbone was recorded on a Nicolet Avatar 320 FT-IR spectrometer (Thermo Fisher
grafted with polyglycerol dendrons of different generations Scientific, Dreieich, Germany). A TSQ 7000 (Finnigan MAT GmbH,
and octadecyl chains in varying proportion, this led to a series Bremen, Germany) instrument was used for ESI measurements,
of new multiamphiphilic polymers that self-assemble into and a JEOL JMS-SX-102A spectrometer (JEOL, Eching, Germany) for
well-defined, globular micelles with diameters in the 10 nm high-resolution mass spectra. Elemental analyses were performed
range at low critical aggregation concentrations (10−5 M), as on a Perkin-Elmer EA 240. Molecular weight and molecular weight
M
was confirmed by DLS, cryo-TEM, and surface tension meas- distribution w/ Mn of polymers were determined using a GPC
urements. The potential ability of the micelles to function as equipped with Agilent 1100 pump, refractive index detector, and
nanocarriers as prospective drug delivery systems was illus- PLgel and Suprema columns. The polymers were eluted with THF
trated with the hydrophobic model dye pyrene. The hydro- at a flow rate of 1.0 mL min−1 . Molecular weights were calibrated
phobic/hydrophilic balance of the multiamphiphiles plays an with pullulan standards. Critical aggregation concentrations of the
important role in guest encapsulation and solubilization. Due polymers were determined via surface tension measurements by
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Non-ionic Dendronized Multiamphiphilic Polymers as Nanocarriers
a commercially available pendant drop tensiometer OCA 20 from after excitation at 320 nm and for ANS binding experiments emis-
Dataphysics, Germany. Aqueous samples in Milli-Q water were pre- sion spectra were recoreded between 400 and 600 nm after excita-
pared 24 h before use and measurements were done at 25 5 °C. tion at 360 nm. Both excitation and emission slit were set at 5 nm.
The aggregation behavior of the multiamphiphiles in water was All data analysis was performed using Microsoft Excel and Sigma
studied over the concentration range of 7 × 10−7 to 1 × 10−3 M. The Plot 8.0 software.
surface tension of each hanging drop was determined two times
ANS Binding Experiments: The binding experiments were car-
per minute and the measurement was stopped when the surface ried out with dendronized multiamphiphile 10f and ANS as the
tension did not change by more than 0.1 mN m−1 over two min- anionic fluorescent probe. Sample solutions were prepared in
utes. Calculations were done using the Young–Laplace equation.
phosphate buffered saline (PBS: 20 mmol L−1 , pH = 7.6). Binding
Light Scattering Measurements: Dynamic light scattering meas- constant (Kb) and number of binding centers per aggregate (n)
urements were carried out on a Zetasizer Nano ZS analyzer with were determined by a double fluorometric titration technique and
integrated 4 mW He–Ne laser, λ = 633 nm (Malvern Instruments calculated using Scatchard-Klotz analysis.[35,37] For the first fluoro-
Ltd, Worchestershire, UK), using backscattering detection (scat- metric titration, increasing concentrations of polymers were added
tering angle θ = 173°) with an avalanche photodiode detector. to a solution of constant ANS concentration (C1ANS). The maximum
The Zetasizer is equipped with a thermostated sample chamber intensity of ANS fluorescence (Fmax), indicating complete binding
controlled by a thermoelectric peltier. The respective polymer was of ANS to the micelles, was then determined. The specific fluores-
dissolved in Milli-Q water and vigorously stirred for at least 12 h cence intensity (Fsp) for bound ANS was calculated as Fmax/C1
.
ANS
at room temperature. The mixture was filtrated via 0.45 μm poly- In the second fluorometric titration, the fluorescence intensity (F)
tetrafluorethylene (PTFE) filters and allowed to equilibrate for 6 h in the reversed system (increasing concentrations of ANS and a
at room temperature. Disposable UV transparent cuvettes (12.5 × constant concentration of the polymer (Cpolym)) was measured.
12.5 × 45 mm, Sarstedt AG & Co, Nümbrecht, Germany) were used The concentration of ANS bound (Cbound ANS) was calculated as
for all measurements.
F/Fsp and the concentration of free ANS (CfreeANS) was calculated
against 1/Cfree
and
Cryogenic TEM: Droplets of the sample solutions were applied as (C2ANS–CboundANS). Plots of C10f/ Cbound
ANS
ANS
to perforated (1 μm hole diameter) carbon film-covered 200 mesh linear regression gave the values for Kb and n according to the fol-
grids (R1/4 batch of Quantifoil MicroTools GmbH, Jena, Germany), lowing equation:
which had been hydrophilized before use by 60 s plasma treat-
ment at 8 W in a BALTEC MED 020 device. The supernatant fluid
C10f / C AbNouSnd = [1/(K b × n × C AfrNeeS )] + (1/n)
was removed with a filter paper until an ultrathin layer of the
sample solution was obtained spanning the holes of the carbon
film. The samples were immediately vitrified by propelling the grids
into liquid ethane at its freezing point (90 K) with a guillotine-like
plunging device. The vitrified samples were subsequently trans-
ferred under liquid nitrogen into a Philips CM12 TEM (FEI, Oregon)
Supporting Information
using a Gatan (Gatan, Inc., California) cryoholder and stage (model
626). Microscopy was carried out at a 94 K sample temperature
using the low-dose protocol of the microscope at a primary mag-
Supporting Information is available from the Wiley Online Library
or from the author.
nification of 58 300× and an accelerating voltage of 100 kV (LaB6-
illumination) with the defocus set to 1.8 μm.
Acknowledgements
Absorbance Measurements: Absorption spectra were recorded
between 220 and 800 nm using a Scinco S-3150 UV–vis spectro-
photometer (Scinco Co. Ltd., Seoul, Korea) (range: 187–1193 nm;
We gratefully acknowledge the financial assistance by the Center for
Supramolecular Interactions (CSI) within the focus area Nanoscale
resolution: 1024 points). All measurements were carried out in a
at the Freie Universität Berlin, the Deutsche Forschungsgemein-
thermostated UV cell (1 cm).
schaft DFG, and Indo-German Science & Technology Centre (IGSTC)
Pyrene Encapsulation Experiment: An excess amount of pyrene
supported by the Bundesministerium für Bildung und Forschung
was added in solid state to aqueous polymer solutions of fixed
(BMBF) and Departments of Science & Technology (DST), Delhi.
concentrations. After stirring for 12 h, the mixture was filtrated
twice via 0.45 μm pore size PTFE syringe filters to remove insoluble
excess of pyrene. The clear polymer solutions were freeze-dried
and re-dissolved in chloroform. The chloroform solutions were
then analyzed by means of UV–vis spectroscopy. The molar extinc-
[1] a) R. Duncan, Nat. Rev. Drug Discov. 2003, 2, 347–360;
b) R. Duncan, R. Gaspar, Mol. Pharmaceu. 2011, 8, 2101–2141.
[2] a) M. Calderon, M. A. Quadir, S. K. Sharma, R. Haag, Adv. Mater.
tion coefficient (εmax) was calculated from a calibration curve of
pyrene in chloroform at λ = 329 nm and used for the concentration
measurements.
Fluorescence Measurements: Fluorescence emission spectra
were taken with a Jasco FP-6500 spectrofluorimeter equipped with
a thermostated cell holder, a DC powered 150 W Xenon lamp, a
Hamamatsu R928 photomultiplier (Hamamatsu Photonics UK Lim-
ited, Essex, UK), and a variable slit system. For pyrene-copolymer
solutions emission spectra were recorded between 350 and 500 nm
2010, 22, 190–218; b) A. Mohr, R. Haag in Applications of
Supramolecular Chemistry: Supramolecular Drug Delivery Sys-
tems (Ed: H.-J. Schneider), Taylor & Francis, CRC Press, Boca
Raton, FL 2012, pp.341–362.
[3] K. Miyata, N. Nishiyama, K. Kataoka, Chem. Soc. Rev. 2012, 41,
2562–2574.
[4] N. Kamaly, Z. Xiao, P. M. Valencia, A. F. Radovic-Moreno,
O. C. Farokhzad, Chem. Soc. Rev. 2012, 41, 2971–3010.
[5] E. R. Gillies, J. M. J. Fréchet, Drug Discov. Today 2005, 10,
35–43.
www.small-journal.com
903
small 2013, 9, No. 6, 894–904
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

S. Gupta et al.
full papers
[6] S. Gupta, R. Tyagi, V. S. Parmar, S. K. Sharma, R. Haag, Polymer [18] K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Angew.
2012, 53, 3053–3078. Chem. Int. Ed. 2010, 49, 6288–6308.
[7] a) J. Khandare, M. Calderon, N. M. Dagia, R. Haag, Chem. Soc. [19] B. Obermeier, F. Wurm, C. Mangold, H. Frey, Angew. Chem. Int.
Rev. 2012, 41, 2824–2848; b) R. Haag, F. Kratz, Angew. Chem. Ed. 2011, 50, 7988–7997.
2006, 118, 1218–1237; Angew. Chem. Int. Ed. 2006, 45, [20] O. V. Borisov, A. Halperin, Langmuir 1995, 11, 2911–2919.
1198–1215.
[21] A. Halldorsson, C. D. Magnusson, G. G. Haraldsson, Tetrahedron
2003, 59, 9101–9109.
[22] K. Faber, Biotransformations in Organic Chemistry, 5th ed.,
Springer Verlag, Berlin 2004; K. Drauz, H. Waldmann, Enzyme
Catalysis in Organic Synthesis, Vol 1,2, VCH, Weinheim, Germany
1994.
[8] H. Frauenrath, Prog. Polym. Sci. 2005, 30, 325–384.
[9] A.-D. Schlüter, Topics Curr. Chem. 1998, 197, 165–191.
[10] a) D. A. Tomalia, P. M. Kirchhoff, Rod-shaped dendrimers, 694.
Midland, MI, USA: The Dow Chemical Company, US Patent
4,694,064, 1987; b) D. A. Tomalia, A. M. Naylor, W. A. Goddard
III, Angew. Chem. 1990, 102, 119–157; Angew. Chem. Int. Ed. [23] a) R. Kumar, M.-H. Chen, V. S. Parmar, L. A. Samuelson, J. Kumar,
Engl. 1990, 29, 138–175; c) R. Yin, Y. Zhu, D. A. Tomalia, H. Ibuki,
J. Am. Chem. Soc. 1998, 120, 2678–2679.
[11] C. J. Hawker, J. M. J. Fréchet, Polymer 1992, 33, 1507–1511.
[12] a) V. Percec, M. Lee, J. Heck, H. E. Blackwell, G. Ungar,
R. Nicolosi, S. Yoganathan, A. C. Watterson, J. Am. Chem. Soc.
2004, 126, 10640–10644; b) S. K. Sharma, M. Husain, R. Kumar,
L. A. Samuelson, J. Kumar, A. C. Watterson, V. S. Parmar, Pure
Appl. Chem. 2005, 77, 209–226.
A. Alvarez-Castillo, J. Mater. Chem. 1992, 2, 931–938; [24] G. Godeau, C. Staedel, P. Barthelemy, J. Med. Chem. 2008, 51,
b) V. Percec, J. Heck, M. Lee, G. Ungar, A. Alvarez-Castillo, J. Mater. 4374–4376.
Chem. 1992, 2, 1033–1039; c) V. Percec, J. Heck, D. Tomazos, [25] S. Malhotra, M. Calderon, A. K. Prasad, V. S. Parmar, R. Haag,
F. Falkenberg, H. Blackwell, G. Ungar, J. Chem. Soc. Perkin Trans.
1993, 1, 2799–2811.
[13] a) A.-D. Schlüter, J. P. Rabe, Angew. Chem. Int. Ed. 2000, 39, 864–
Org. Biomol. Chem. 2010, 8, 2228–2237.
[26] I. Astafieva, X. F. Zhong, A. Eisenberg, Macromolecules 1993, 26,
7339–7352.
883; b) A. Zhang, L. Shu, Z. Bo, A.-D. Schlüter, Macromol. Chem. [27] C. Cheng, M. Schmidt, A. Zhang, A.-D. Schlüter, Macromolecules
Phys. 2003, 204, 328–339; c) R. Freudenberger, W. Claussen, 2007, 40, 220–227.
A.-D. Schlüter, H. Wallmeier, Polymer 1994, 35, 4496–4501; [28] D. Xie, M. Jiang, G. Zhang, D. Chen, Chem. Eur. J. 2007, 13,
d) B. Zhang, A.-D. Schlüter, New J. Chem. 2012, 36, 414–418. 3346–3353.
[14] J. I. Paez, M. Martinelli, V. Brunetti, M. C. Strumia, Polymers 2012, [29] A. Halperin, Macromolecules 1991, 24, 1418–1419.
4, 355–395.
[ 3 0 ] F . T a n a k a , J. Non-Cryst. Solids 2002, 307, 688–697.
[31] K. T. Oh, Y. T. Oh, N.-M. Oh, K. Kim, D. H. Lee, E. S. Lee, Int. J.
Pharm. 2009, 375, 163–169.
[15] Y. Chen, X. Xiong, Chem. Commun. 2010, 46, 5049–5060.
[16] a) S. Gupta, M. K. Pandey, K. Levon, R. Haag, A. C. Watterson,
V. S. Parmar, S. K. Sharma, Macromol. Chem. Phys. 2010, 211, [32] Z. Sideratou, D. Tsiourvas, C. M. Paleos, J. Colloid Interface Sci.
239–244; b) S. Kumar, A. Mohr, A. Kumar, S. K. Sharma, R. Haag,
Int. J. Artif. Organs 2011, 34, 84–92.
2001, 242, 272–276.
[33] K. Kalyanasundaram, J. K. Thomas, J. Am. Chem. Soc. 1977, 99,
[17] a) M. Wyszogrodzka, K. Möws, S. Kamlage, J. Wodzinska,
2039–2044.
B. Plietker, R. Haag, Eur.
b) M. Wyszogrodzka, R. Haag, Chem. Eur. J. 2008, 14, 9202–9214;
c) I. N. Kurniasih, H. Liang, J. P. Rabe, R. Haag, Macromol. Rapid [35] a) D. Shcharbin, B. Klajnert, V. Mazhul, M. Bryszewska,
J
Org. Chem. 2008, 53–63; [34] R. K. Kainthan, C. Mugabe, H. M. Burt, D. E. Brooks, Biomacro-
molecules 2008, 9, 886–895.
Commun. 2010, 31, 1516–1520; d) I. N. Kurniasih, H. Liang,
V. D. Moeschwitzer, M. A. Quadir, M. Radowski, J. P. Rabe, R. Haag,
New J. Chem. 2012, 36, 371–379; e) B. Trappmann, K. Ludwig,
J. Fluoresc. 2003, 13, 519–524; b) M. Jokiel, D. Shcharbin,
J. Janiszewska, Z. Urbanczyk-Lipkowska, M. Bryszewska, J. Fluo-
resc. 2007, 17, 73–79.
M. R. Radowski, A. Shukla, A. Mohr, H. Rehage, C. Böttcher, [36] J. Slavik, Biochim. Biophys. Acta 1982, 694, 1–25.
R. Haag, J. Am. Chem. Soc. 2010, 132, 11119–11124; f) H. Türk, [37] G. Scatchard, Ann. NY Acad. Sci. 1949, 51, 660–672.
A. Shukla, P. C. Alves Rodrigues, H. Rehage, R. Haag, Chem.
Eur. J. 2007, 13, 4187–4196; g) C. S. Popeney, M. C. Lukowiak,
C. Böttcher, B. Schade, P. Welker, D. Mangoldt, G. Gunkel,
Z. Guan, R. Haag, ACS Macro Lett. 2012, 1, 564–567.
Received: June 5, 2012
Revised: September 3, 2012
Published online: December 6, 2012
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