A pH-responsive superamphiphile based on dynamic covalent bonds

Article abstract of DOI:10.1002/chem.201003502

Under pH control: A successful example of a toothbrush-type superamphiphile based on dynamic covalent bonds is demonstrated. The superamphiphile can self-assemble in water to form spherical aggregates, which disassemble reversibly under mildly acidic cond

Full text of DOI:10.1002/chem.201003502

DOI: 10.1002/chem.201003502
A pH-Responsive Superamphiphile Based on Dynamic Covalent Bonds
[
a]
Chao Wang, Guangtong Wang, Zhiqiang Wang, and Xi Zhang*
Stimuli-responsive polymers have made rapid advances in
recent years because of prospective applications in biotech-
[1]
nology and drug-delivery systems. Conventionally, the
stimuli-responsive groups are linked to polymers by covalent
synthesis. The new concept of “superamphiphiles” has
emerged as an alternative and powerful method for fabricat-
ing stimuli-responsive self-assembled structures. Superam-
phiphiles refers to amphiphiles that are synthesized by non-
[2]
covalent interactions. Thus, in superamphiphiles, stimuli-
responsive moieties can be linked to amphiphiles on the
basis of noncovalent interactions that greatly reduces the
[3–4]
need for tedious chemical synthesis.
A dynamic covalent bond is in many aspects like a nonco-
valent interaction due to the dynamic nature and has al-
ready led to controlled formation of gels, homopolymers,
[5]
functional surfaces, and so forth. Recently, van Esch et al.
reported, for the first time, a low-molecular-weight amphi-
[5g]
phile on the basis of dynamic covalent bonds. Among dy-
[6]
namic covalent bonds, the benzoic imine bond is especially
attractive, because whereas it is stable under physiological
conditions, the linker can be hydrolyzed under mildly acidic
[7]
conditions close to the extracellular pH of solid tumors.
Scheme 1. Schematic representation of the building blocks of the super-
amphiphile and the pH-responsive property of the self-assembled aggre-
gates. The superamphiphile can self-assemble into spherical aggregates,
which disassemble when the pH is decreased to 6.5.
Although dynamic covalent bonding has been used widely
in the controlled formation of various supramolecular sys-
tems, the usefulness as a driving force for fabrication of
polymeric superamphiphiles has not been reported. Herein
we report the successful synthesis of a toothbrush-type,
polymeric superamphiphile based on dynamic benzoic imine
bonds by using a double-hydrophilic polymer with amine
moieties and an organic molecule with benzoic aldehyde
groups as building blocks. The superamphiphile can self-as-
semble into spherical aggregates under physiological condi-
tions. Due to the unique pH-responsive nature of the benzo-
ic imine bond, the superamphiphile can be formed and dis-
sociated reversibly in response to small pH fluctuations in
the physiologically accessible range; this leads to self-assem-
bly and disassembly of the aggregates.
double-hydrophilic block copolymer, methoxy-poly(ethy-
leneglycol)114–block–poly(l-lysine hydrochloride)200 (PEG–
b–PLKC) in which the PLKC segment contains primary
amine groups. The other component is 4-(decyloxy)benzal-
dehyde (DBA), which contains an alkyl chain and a benzal-
dehyde end group. Under physiological conditions (pH 7.4),
the alkyl chain in DBA can be attached to the polylysine
segment by benzoic imine bonds formed between the amine
groups on the polylysine side chain and benzaldehyde
groups in DBA; this leads to the formation of a toothbrush-
type superamphiphile. When the pH is reduced to 6.5, how-
ever, the benzoic imine bond is broken and the PEG–b–
PLKC/DBA superamphiphile decomposes into nonamphi-
philic components, resulting in disassembly of the aggregates
and release of guest molecules. When the pH changes back
to 7.4, the benzoic imine bond is retrieved and the aggrega-
tion is recovered.
The toothbrush-type superamphiphile consists of two
components. As shown in Scheme 1, one component is a
[
a] C. Wang, G. Wang, Prof. Z. Wang, Prof. X. Zhang
Key Lab of Organic Optoelectronics and Molecular Engineering
Department of Chemistry, Tsinghua University
Beijing 100084 (P.R. China)
Fax : (+86)10-62771149
To prepare the superamphiphile, PEG–b–PLKC and
DBA were mixed in a phosphate buffer, pH 7.4, at the
molar ratio PEG–b–PLKC:DBA 1:200 to ensure that there
E-mail: xi@mail.tsinghua.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003502.
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Chem. Eur. J. 2011, 17, 3322 – 3325

COMMUNICATION
were equimolar amine and benzaldehyde groups. After soni-
cation for 10 min, a transparent aqueous solution was ob-
tained. Because the solubility of DBA is rather low in
water, the enhanced solubility is attributed to the formation
of benzoic imine bonds. The chemical structure of the super-
amphiphile was confirmed by NMR spectroscopy. As shown
in Figure 1, the NMR signals for the aldehyde groups disap-
the increase of aldehyde concentration. As a result, the su-
peramphiphile with an amine:aldehyde ratio of 1:1, with a
critical micelle concentration (cmc) of 0.012 mgmL , was
À1
chosen as a model system for investigation.
The concentration of the sample solution of the superam-
À1
phiphile was 0.043 mgmL , about three times higher than
the cmc, to ensure the formation of self-assembled polymer
micelles. The self-assembly behavior of the toothbrush-like
superamphiphile was investigated by TEM and dynamic
laser scattering (DLS).
An aqueous solution of the superamphiphile with a PEG–
À1
b–PLKC concentration of 0.75 mgmL was prepared in
phosphate buffer, pH 7.4. The TEM image in Figure 2a re-
1
Figure 1. The H NMR spectrum (D
2
O) of PEG–b–PLKC, DBA, and the
mixture of PEG–b–PLKC and DBA in the molar ratio of 1:200 at
pH 7.4.
pear. Because the molar ratio between amine groups and
benzoic aldehyde is 1:1, the absence of the aldehyde proton
signal indicates that nearly all of the aldehyde groups were
converted. A new signal appeared at 6.5 ppm, corresponding
to the proton on the benzoic imine conjugate structure. At
the same time, the signals of the protons on the alkyl chain
of DBA merge into two broad signals near 0.5–2 ppm; this
is a typical feature of protons on polymers. Further evidence
of the formation of the superamphiphile is provided by
FTIR. As shown in the Supporting Information, the charac-
Figure 2. a) TEM images of PEG–b–PLKC/DBA aggregates in a pH 7.4
buffer. b) The statistical analysis of the diameter of spherical aggregates.
c) DLS of PEG–b–PLKC/DBA complex at pH 7.4. d) TEM image of
PEG–b–PLKC/DBA at pH 6.5.
veals that the superamphiphile self-assembled into spherical
micelles in water. The average diameter of the aggregates
according to the TEM results is about 65 nm (Figure 2b).
The size of the polymer micelles was further studied by
DLS, which gave an average diameter of 70 nm. At pH 6.5,
the TEM images showed that very few aggregates were
present in the solution, thus indicating disassembly of the
aggregates (Figure 2d).
NMR and FTIR experiments were performed to confirm
the pH responsiveness of the superamphiphile. Figure 3a
shows at pH was changed to 6.5, the signal at 7.8 ppm corre-
sponding to the imine bond disappeared accompanied by
the appearance of a sharp signal at 9.5 ppm, which corre-
sponds to the aldehyde proton. Simultaneously, the broad
signals between 0.5 and 2 ppm separated into a few sharp
signals, confirming the decomposition of the superamphi-
phile. When the pH was changed back to pH 7.4, the imine
signal was recovered and the broad signals at 0.5–2 ppm re-
appeared, confirming that the formation and decomposition
of the superamphiphile is reversible. In the FTIR spectra
(see the Supporting Information) the absorption band at
À1
teristic IR band of the imine at 1650 cm is clearly visible
in the spectrum of the PEG–b– PLKC/DBA complex.
The influence of the amine:aldehyde ratio was evaluated.
When the concentration of PEG–b–PLKC was kept con-
À1
stant at 0.019 mgmL , samples of superamphiphiles with
different amine:aldehyde ratios were prepared. As shown in
Figure S3 in the Supporting Information, when the molar
ratio is 10:1, 5:1, 3:1, 2:1, the count rates are very low, indi-
cating that no aggregates are formed. When the amine:alde-
hyde ratio is 1:1 and all the amine groups have been con-
verted, the count rate increases to 120 kilocounts per second
(
kcps) instantly, indicating the formation of micellar aggre-
gates. The formation of aggregates can be explained by the
growth of the hydrophobic part. Upon addition of DBA, the
hydrophilic protonated amine groups are gradually convert-
ed into hydrophobic alkyl chains. Therefore, the double-hy-
drophilic polymer changes into polymeric amphiphiles, in-
ducing the formation of aggregates. The increase of the hy-
drophobic part can also be confirmed by using pyrene as a
florescence probe. The I /I value of pyrene decreases upon
1
3
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www.chemeurj.org
3323

X. Zhang et al.
Figure 4. a) The fluorescence intensity of NR at different pH values.
b) The controlled-release capability of PEG–b–PLKC/DBA determined
through the time dependence of the fluorescence intensity of NR.
the amount of encapsulated NR determined by fluorescence
emission microscopy. Above pH 7.4 the amount of NR en-
capsulated was almost constant at a concentration of about
À1
0
.6 mgmL according to the fluorescence intensity. Howev-
1
Figure 3. a) H NMR spectra of PEG–b–PLKC/DBA at different pH
er, at a mildly acidic pH value (6.5), the fluorescence inten-
sity decreased drastically and the encapsulated concentra-
tion dropped to about 0.05 mgmL , indicating release of the
values: 7.4, 6.5, and again 7.4. b) The zeta potential of the PEG–b–
PLKC/DBA aggregates at different pH values. c) The change of the zeta
potential in the pH-tuning process.
À1
guest molecules. The release kinetics was studied by time-
dependent fluorescence microscopy. Figure 4b shows that
when the pH changed from 7.4 to 6.5, the encapsulated NR
was released in less than 20 min, reflecting the fast-release
feature of the self-assemblies. At the same time, NR can be
used as a hydrophobic probe. As shown in the Supporting
Information, in a solution of PEG–b–PLKC/DBA in phos-
phate buffer (pH 7.4), NR emission was observed at 641 nm,
that is, emission in a hydrophobic environment. At pH 6.5,
the wavelength of the maximum emission gradually shifted
to 661 nm, which is the typical emission wavelength of NR
in water. The redshift of the NR emission further confirms
the disassembly of the aggregates and release of the guest
molecules upon pH stimulus.
In conclusion, we have reported the first example of a
polymeric superamphiphile based on dynamic covalent
bonds. The superamphiphile can self-assemble in water to
form spherical polymer micelles under physiological condi-
tions and the aggregates disassemble in response to a pH
stimulus, thus providing a new carrier for loading and releas-
ing guest molecules. It should be noted that the assembly
and disassembly processes are reversible and that disassem-
bly occurs at pH 6.5, near the extracellular pH of tumor
cells. Moreover, the loaded guest molecules can be rapidly
released. We anticipate that dynamic covalent bonding can
be used as a driving force to fabricate superamphiphiles
with different architectures as an approach to new intelli-
gent materials.
À1
1
650 cm , ascribed to the stretching vibration of carbon–ni-
trogen double bond, was significantly weakened when the
pH was reduced to 6.5. The intensity of the 1650 cm band
increased when the pH was changed back to 7.4.
À1
Because the PEG–b–PLKC/DBA complex is pH respon-
sive, we considered that the pH responsiveness is responsi-
ble for the disassembly of the spherical aggregates shown in
Figure 2d. To provide further evidence, the aggregation be-
havior at pH 6.5 was also monitored by DLS count rate,
which revealed that the aggregates disappeared at pH 6.5.
When the pH was changed back to 7.4, TEM images
showed that the spherical aggregates were formed again,
and DLS gave an average diameter of 70 nm, which indi-
cates that the pH-responsive self-assembly and disassembly
processes are indeed reversible in accordance with the rever-
sible pH responsiveness of the formation and decomposition
of the superamphiphile.
The self-assembly and disassembly process in response to
pH was also monitored by zeta potential measurements. Be-
cause the pKa of PLKC is about 9.0, the aggregates re-
mained positively charged in the pH region of interest. As
shown in Figure 3b, above pH 7.4 the zeta potential was
close to zero as a result of the high conversion of amino
groups and the shielding effect of PEG. When the pH was
reduced to 6.5 a significant increase of the zeta potential
was observed, indicating decomposition of the superamphi-
phile and disassembly of the aggregates. When the pH was
changed back to 7.4 the zeta potential returned to zero
again (Figure 3c), providing additional confirmation of the
reversible nature of the self-assembly.
Acknowledgements
The PEG–b–PLKC/DBA aggregate was further studied as
a possible medium for encapsulation and release of guest
molecules in response to pH fluctuations under physiologi-
cal conditions. Nile Red (NR) as a model guest molecule
was loaded into the spherical aggregates. Figure 4a shows
This work was financially supported by the National Basic Research Pro-
gram (2007CB808000), NSFC (50973051, 20974059), NSFC–DFG joint
grant (TRR 61) and Tsinghua University Initiative Scientific Research
Program (2009THZ02230). We acknowledge Prof. Lidong Li and Fu
Tang at the University of Science and Technology Beijing for help with
the DLS experiments.
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Chem. Eur. J. 2011, 17, 3322 – 3325

A pH-Responsive Superamphiphile
COMMUNICATION
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Keywords: amphiphiles · benzoic imine · dynamic covalent
bonds · pH responsive · self-assembly
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Received: December 5, 2010
Published online: February 21, 2011
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3325