A versatile and robust vesicle based on a photocleavable surfactant for two-photon-tuned release

Article abstract of DOI:10.1002/chem.201300526

A small amphiphile that contains a coumarin unit and alkynyl groups, as a two-cleavable segment and polymerizable groups, respectively, was designed and synthesized. The amphiphile showed a critical aggregation concentration of about 4.6×10^-5M and formed a vesicle-type assembly. The formed vesicles were stabilized by insitu "click" polymerization without altering their morphology. Hydrophobic and hydrophilic guests can be encapsulated within the vesicle membrane and inside the aqueous core of the vesicle, respectively. The loaded guests can be released from the vesicle by using UV or near-IR stimuli, through splitting up the amphiphilic structure of the amphiphile. Distinguished dose-controlled photorelease of the polymeric vesicle is achieved with the maintenance of vesicular integrity, which makes the guest release dependent on the amount of cleavage of the amphiphilic structure during irradiation. This study provides a potential strategy for the development of versatile and stable drug-delivery systems that offer sustained and photo-triggered release. Copyright

Full text of DOI:10.1002/chem.201300526

FULL PAPER
DOI: 10.1002/chem.201300526
A Versatile and Robust Vesicle Based on a Photocleavable Surfactant for
Two-Photon-Tuned Release
Jianming Dong,[a] Zhiqing Xun,[a] Yi Zeng,*[a] Tianjun Yu,[a] Yongbin Han,[a]
Jinping Chen,[a] Ying-Ying Li,[a] Guoqiang Yang,*[b] and Yi Li*[a]
Abstract: A small amphiphile that con-
tains coumarin unit and alkynyl
be encapsulated within the vesicle
membrane and inside the aqueous core
of the vesicle, respectively. The loaded
guests can be released from the vesicle
by using UV or near-IR stimuli,
through splitting up the amphiphilic
structure of the amphiphile. Distin-
guished dose-controlled photorelease
of the polymeric vesicle is achieved
with the maintenance of vesicular in-
tegrity, which makes the guest release
dependent on the amount of cleavage
of the amphiphilic structure during ir-
radiation. This study provides a poten-
tial strategy for the development of
versatile and stable drug-delivery sys-
tems that offer sustained and photo-
triggered release.
a
groups, as a two-photon-cleavable seg-
ment and polymerizable groups, re-
spectively, was designed and synthe-
sized. The amphiphile showed a critical
aggregation concentration of about
4.6ꢀ10À5 m and formed a vesicle-type
assembly. The formed vesicles were sta-
bilized by in situ “click” polymerization
without altering their morphology. Hy-
drophobic and hydrophilic guests can
Keywords: amphiphiles · controlled
release · drug delivery · host–guest
systems · vesicles
Introduction
led-release function. However, most of the phototriggered-
release systems that have been reported to date employ a
single-photon stimulus, such as UV or visible light, which is
easily absorbed by tissues and may cause damage to normal
cells. Near-infrared (NIR) stimuli within the range of the
optical window in tissues (approximately 700–1000 nm), in
which the absorption and scattering by tissues are low,
enable deeper penetration and less risk of damage. If the
stimulus can be initiated by excitation with two or more co-
incident NIR photons with the equivalent total energy of a
single photon of UV or visible light, such phototriggered re-
lease would become a more-promising approach for biologi-
cal applications. Furthermore, the two-photon absorption of
a photolabile unit is quadratically dependent on the intensi-
ty of the light, thus providing exceptional spatial precision
by confining the photolysis to the focal spot of the stimulus
laser.[20] Although several two-photon-responsive aggregates
that are composed of polymeric or dendritic molecules have
recently been reported,[21–24] the development of regulated
two-photon-release carriers is still an appealing field for fur-
ther exploration.
Compared to various nanocarriers of macromolecules and
inorganic materials that show stimuli-responsive release, as-
semblies of small amphiphiles, such as micelles, vesicles, lip-
osomes, and capsules, remain active and attractive in indus-
trial and scientific areas, owing to their ease of synthesis, bi-
ocompatibility, diverse architectures, and functional versatil-
ity.[25,26] However, the application of surfactant assemblies in
drug delivery is hampered by their insufficient thermody-
namic stability and susceptibility to dilution and complex
environments in biological systems, which usually lead to
the collapse of the assemblies and the premature release of
Over the last decade, great effort has been devoted to the
development of drug-delivery systems for therapeutic appli-
cations.[1–3] Various kinds of inorganic and organic assem-
blies that are capable of the encapsulation and controlled
release of guest molecules, triggered by internal or external
stimuli, such as pH value,[4,5] temperature,[6–8] enzyme,[9–12]
redox,[13–15] and light,[16–19] have been developed. The use of
light to trigger the release of a guest is one of the most-at-
tractive fields currently being investigated owing to the ad-
vantages of remote, temporal, and spatial control. Photores-
ponsive properties have been incorporated into different
kinds of drug-delivery carriers, such as micelles, vesicles, and
nanoparticles, thereby effectively achieving a photocontrol-
[a] Dr. J. Dong, Z. Xun, Dr. Y. Zeng, Dr. T. Yu, Dr. Y. Han, Dr. J. Chen,
Dr. Y.-Y. Li, Prof. Dr. Y. Li
Key Laboratory of Photochemical Conversion and
Optoelectronic Materials
Technical Institute of Physics and Chemistry
Chinese Academy of Sciences
Beijing 100190 (P.R. China)
Fax : (+86)10-82543518
E-mail: zengyi@mail.ipc.ac.cn
yili@mail.ipc.ac.cn
[b] Prof. Dr. G. Yang
Beijing National Laboratory for Molecular Sciences (BNLMS)
Key Laboratory of Photochemistry
Institute of Chemistry, Chinese Academy of Sciences
Beijing 100190 (P.R. China)
E-mail: gqyang@iccas.ac.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300526.
Chem. Eur. J. 2013, 19, 7931 – 7936
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7931

the payloads. One of the practical routes for improving the
instability of an assembly is to polymerize the assemblies
through the proper design and modification of the amphi-
philes.[27–29] Besides the challenge of stability, the develop-
ment of surfactant vehicles with dose-controlled release
holds promise for drug delivery and other biological applica-
tions. Most stimuli-responsive carriers undergo dissociation
once they exposed to stimuli, thereby resulting in the
“burst” release of encapsulated guests. Until now, not many
examples of drug-delivery vehicles that offer stepwise or
sustained release have been demonstrated.[30–32] Herein, we
report the synthesis of robust and nanosized vesicles, which
are formed from a new surfactant, with a highly controlled
release function that is triggered by NIR light.
sembly through efficient “click” reactions with appropriate
multiazido molecules. It was expected that the stabilized as-
sembly of amphiphile 1 can act as a versatile, controlled-re-
lease vehicle for hydrophobic and hydrophilic cargoes
through the disruption of the amphiphilic structure by a
NIR trigger (Scheme 1).
The assembly behavior of amphiphile 1 was investigated
in water by using Nile Red (NR) as a hydrophobic fluores-
cent probe. The change in the emission intensity of NR,
which corresponded to various concentrations of amphiphile
1, was used to monitor the formation of the aggregate, thus
giving a critical aggregation concentration (CAC) of 4.6ꢀ
10À5 m, which was consistent with the results of concentra-
tion-dependent conductivity measurements (4.4ꢀ10À5 m; see
the Supporting Information, Figure S2). Above its CAC, am-
phiphile 1 formed aggregates with an average hydrodynamic
diameter of 180 nm, as given by dynamic light scattering
(DLS) measurements (see the Supporting Information, Fig-
ure S3a). Transmission electron microscopy was used to
reveal the morphology of the aggregates that were formed
by amphiphile 1, as shown in Figure 1a. These aggregates
Results and Discussion
The surfactant was designed and synthesized by using a cou-
marin derivative as the two-photon-cleavable unit to con-
nect the hydrophobic tails of two tetradecyl groups and a
hydrophilic alkynylated ammonium head-group. A 7-amino-
coumarin-4-ylmethyl ester was selected as the photolabile
group, owing to its sufficient two-photon-absorption cross-
section and mature application in caged compounds.[20,33]
The coumarin unit was alkylated with n-tetradecyl bromide
and then coupled with dipropargylaminoethanol, which was
finally converted into the ammonium moiety with bromo-
ethane, thus giving the target cationic amphiphile (1,
Scheme 1; for detailed synthesis and characterization data,
Figure 1. TEM images of vesicles that were formed from amphiphile 1
a) before and b) after the “click” reactions. Inset shows a magnified pho-
tograph of a single vesicle.
showed a spherical shape, with an obvious contrast between
the periphery and the central area of the sphere, which is in-
dicative of the formation of vesicles in aqueous solution.
The vesicular structure was further confirmed by observing
the encapsulation of a hydrophilic guest, Rhodamine 6G
(Rh6G), inside the interior of the spherical assembly, which
denoted the existence of an aqueous phase inside the aggre-
gates (see the Supporting Information, Figure S5).
Scheme 1. Photolysis of amphiphile 1 (top) and the versatile vesicle that
was formed from amphiphile 1 for the controlled release of hydrophilic
or hydrophobic guests (bottom).
As mentioned above, one disadvantage that is commonly
encountered in the application of surfactant aggregates as
drug carriers is their instability and their polymerization is
considered to be a feasible way to overcome this problem.
The “click” reaction between an alkyne and an azide has
been demonstrated to be one of the most-effective ways to
modify aggregates under mild conditions[29] and we used it
herein to stabilize the vesicles. Thus, the alkynyl groups on
the membrane surface of the vesicles were treated with 1,2-
bis(2-azidoethoxy)ethane, a water-soluble diazido linker, in
the presence of CuCl2 and sodium ascorbate at room tem-
see the Supporting Information). Amphiphile 1 contains an
alkynyl ammonium group as a large hydrophilic head-group
and two fluid n-tetradecyl chains as hydrophobic groups,
which induce a tendency to form vesicle-type assemblies in
solution.[34] Upon single- or two-photon excitation, the pho-
tolysis of the coumarin ester will lead to the cleavage of am-
phiphile 1. Amphiphile 1 self-assembles in water with the al-
kynyl groups arranging on the membrane of the vesicles,
which could be used to stabilize or post-functionalize the as-
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Photocleavable Surfactant for Two-Photon-Tuned Release
FULL PAPER
perature, thereby affording the polymerized vesicles. DLS
analysis indicated that the click reaction did not notably
alter the size of the vesicles (see the Supporting Informa-
tion, Figure S3a). TEM analysis further confirmed the reten-
tion of vesicular structure after the click reaction, as shown
in Figure 1b. On diluting the aqueous solution of the vesi-
cles to a concentration below the CAC, the polymerized
vesicles still existed, according to the DLS experiments (see
the Supporting Information, Figure S3b), whilst the unpoly-
merized vesicles disassembled, thus indicating that the sta-
bility of the vesicles had been improved by the click poly-
merization.
The photocontrolled release behaviors of a hydrophobic
payload from polymerized and unpolymerized vesicles were
assessed by using NR as a model compound. The encapsula-
tion and release processes of NR by the vesicles were stud-
ied by observing the absorption of NR in the vesicle solu-
tion, because NR was readily solubilized in the vesicle mem-
brane but was insoluble in water. First, the phototriggered
release of NR through a single-photon process was evaluat-
ed upon exposure to UV light (l=365 nm). The absorbance
of NR in the solution of polymerized vesicles decreased
gradually with increasing irradiation time, which indicated
that the photocleavage of amphiphile 1 effectively led to the
release of entrapped NR. The photorelease profiles of the
polymerized and unpolymerized vesicles, as well as of a con-
trol sample (leakage from a polymerized sample in the
dark), are summarized in Figure 2a. The absorption of a sol-
lumination and in the dark. The release of NR from the
polymerized vesicles proceeded under UV irradiation and
ceased once the light had been shut out, thus showing a
stepwise-release curve, as shown in Figure 2b. This result
clearly shows that the dose-controlled release of hydropho-
bic guests can be achieved in the polymerized vesicles by
using a single-photon trigger.
Following the success in single-photon-controlled release,
two-photon-triggered experiments were performed to exam-
ine the release capability of the polymerized/unpolymerized
vesicles by using focused NIR light (800 nm). The absorp-
tion spectra of NR in vesicle solutions were measured as a
function of NIR-exposure time to demonstrate the release
process of NR from the vesicles. The release profiles of the
unpolymerized and polymerized vesicles by using a NIR
trigger, together with a control experiment with the poly-
merized vesicles, are shown in Figure 3. Entrapped NR was
*
Figure 3. a) Release profiles of NR from polymerized ( ) and unpoly-
merized vesicles (&) under NIR illumination and control experiments
~
(
) with the polymerized vesicles. b) Stepwise release of NR from poly-
&
*
merized vesicles in response to intermittent NIR irradiation ( and
in the dark).
=
effectively released from the polymerized/unpolymerized
vesicles under a NIR stimulus as a result of the two-photon
cleavage of amphiphile 1, thus showing analogous kinetics
to the single-photon case. The release of NR from the poly-
merized vesicles was gentler than that from the unpolymer-
ized vesicles, which indicated that the click-stabilized mem-
brane provided a sustainable-release function. The dose-
controlled release character of the polymerized vesicles with
a two-photon stimulus was further evaluated by alternating
the NIR-laser illumination with dark conditions. As expect-
ed, the release kinetics of NR demonstrated a stepwise be-
havior upon intermittent exposure to a NIR laser. The step
height denoted the amount of released NR, which was de-
pendent on the irradiation time. Therefore, we can conclude
that the polymerized vesicles can be used as a dose-control-
led carrier by tuning the two-photon-illumination dose. This
distinct character can be attributed to the polymerization of
the membrane of the vesicles, which maintained their vesic-
ular morphology during the irradiation, as shown by the
DLS results (see the Supporting Information, Figure S4).
This result is further strengthened by the TEM observations
(Figure 4). The main frame of the polymerized vesicles did
not collapse and the unpolymerized vesicles completely
broke-up after irradiation. The polymerization of vesicle
*
Figure 2. a) Release profiles of NR from unpolymerized ( ) and poly-
~
&
merized vesicles ( ) upon UV irradiation and control experiments ( )
with the polymerized vesicle. b) Release of NR in response to periodic
&
*
UV exposure ( ) and control experiments ( =in the dark).
ution of the polymerized vesicles showed no significant
change in the dark. Encapsulated NR was released from of
the polymerized vesicles in a smooth manner during UV ir-
radiation, whilst the NR-release kinetics from unpolymer-
ized vesicles demonstrated a relatively steep trend at the
start of the irradiation and shortly reached a plateau. Taking
account of the photorelease results and the stabilities of dif-
ferent types of vesicles, this sustainable photorelease from
polymerized vesicles can be attributed to the stabilized pe-
riphery of the membrane, which prevents a burst release. In
contrast, the unpolymerized vesicles dissociate quickly
under irradiation, thereby resulting in a rapid release. The
capability of the dose-controlled release was further verified
by periodically subjecting the polymerized vesicles to UV il-
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Y. Zeng, G. Yang, Y. Li et al.
the rate of release can be regulated by adjusting the irradia-
tion time. The release of the hydrophilic guest under irradia-
tion can be ascribed to an increase in the permeability of
the vesicles, caused by the two-photon disruption of the am-
phiphilic structure in the vesicle membrane. The permeabili-
ty of the vesicles determines the release rate of the hydro-
philic guests, which can be adjusted by tuning the irradiation
dose.
Conclusion
Figure 4. TEM images of a) unpolymerized vesicles and b) polymerized
vesicles after NIR irradiation.
In conclusion, we have designed and synthesized a new two-
photon-sensitive amphiphile that contains a photocleavable
coumarin chromophore and two alkynyl units. The vesicles
that are formed by the amphiphile can be polymerized by
using “click” reactions on the vesicular membrane, thereby
resulting in the formation of highly stable nanocarriers.
These stabilized vesicles can incorporate hydrophobic guests
into their membrane and release the payloads upon expo-
sure to UV light (365 nm) or a NIR pulse laser (800 nm)
through photolysis of the coumarin units. The finely regulat-
ed release of the guests was achieved by the polymerized
membrane of the vesicles, which prevented the collapse of
the assembly and the sudden release of the guests during ir-
radiation. Furthermore, the stabilized vesicles can seal hy-
drophilic guests inside the aqueous chamber and release
them upon irradiation, thus implying their use as universal
nanocarriers for multi-area applications. This study provides
the rational and versatile design of stable assemblies of sur-
factants, which show potential for dose-controlled triggered
release in biomedical applications.
membrane avoided the complete collapse of the vesicles
during the photocleavage of amphiphile 1, thus leading to
the residual guest sequestration and irradiation-dose-de-
pendent release kinetics.
Furthermore, the capability of the polymerized vesicles in
the two-photon-stimulated release of hydrophilic guests was
investigated by using Rh6G as the encapsulated guest inside
the aqueous core of the vesicles. Rh6G was added into the
aqueous solution together with amphiphile 1. After assem-
bly, the vesicles were polymerized and sequentially dialyzed
against water to remove the untrapped Rh6G. Aliquots of
the loaded vesicle solution were irradiated with a NIR laser
for different lengths of time and then poured into dialysis
bags, as shown in Figure 5a. The aqueous solution outside
Experimental Section
Methods: IR spectra were recorded on a Varian Excalibur 3100 spec-
trometer. 1H NMR (400 MHz) spectra were recorded on
a Bruker
Avance P-400 spectrometer. MALDI-TOF and ESI mass spectra were
recorded on Bruker MicroFlex and Waters LCT Premier XE mass spec-
trometers, respectively. Milli-Q water (18.2 MWcm) was obtained from a
Milli-Q Integral Ultrapure Water Production Unit. UV/Vis absorption
spectra were recorded on a Shimadzu UV-1601PC spectrometer. Fluores-
cence measurements were performed on a Hitachi F-4500 spectrometer.
Dynamic light scattering measurements were performed on a Malvern
Zetasizer Nano ZS90. Concentration-dependent conductivity was meas-
ured with Shanghai Precision & Instrument DDS-307 conductivity meter.
The TEM samples were negatively stained with uranyl acetate and the
TEM images were recorded on a FEI TECNAI F20 transmission electron
microscope at 200 kV. Single-photon release experiments were performed
by using a mercury lamp that was equipped with a 365 nm band-pass
filter. Two-photon-release examinations were performed with a 1 kHz re-
generative Ti:Sapphire amplifier (Coherent Inc.), which generated
about170 fs pulses at 800 nm. The laser beam was focus onto the sample
cell with a spot size of about 1 mm and an average power of 0.98 W.
Figure 5. a) Schematic representation of the photoinduced release of
Rh6G from the vesicular solution after different exposure times to a NIR
laser. b) Plot of the fluorescence intensity of Rh6G at 550 nm (lex
=
510 nm) from the solution outside the dialysis bag versus different dialy-
~
*
sis times (& =40 and =10 min irradiation; =without irradiation).
the dialysis bag was sampled and examined by monitoring
the fluorescence intensity of Rh6G to estimate the amount
of released guest. A plot of the fluorescence intensity of
Rh6G at 550 nm versus the dialysis time for the vesicles
without and with NIR irradiation for 10 and 40 min is
shown in Figure 5b. The emission intensity shows a steady
increase with dialysis time for the irradiated vesicles and a
negligible change for the unirradiated vesicles, thus indicat-
ing that the release of Rh6G from the vesicles was initiated
by the NIR irradiation. The slope of the release profile in-
creases with the irradiation time, thus demonstrating that
Materials: All of the chemicals were purchased from Alfa Aesar, Al-
drich, or J&K Chemical and used without further purification, unless oth-
erwise stated. Milli-Q water was used in the aqueous experiments.
CH2Cl2 and CHCl3 was distilled from CaH2 and THF was purified by dis-
tillation from sodium with benzophenone.
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Photocleavable Surfactant for Two-Photon-Tuned Release
FULL PAPER
Synthesis and characterization of the compounds: The synthetic route is
shown in the Supporting Information, Scheme S1. The H NMR spectrum
of amphiphile 1 is shown in the Supporting Information, Figure S1.
NCH2), 1.71 (m, 4H; NCH2CH2), 1.55 (t, 3H; NCH2CH3), 1.48–1.28 (m,
1
44H), 0.89 ppm (t, 6H); IR (KBr pellet): n˜ =3265.2, 2921.7, 2851.5,
1739.4, 1709.9, 1607.6, 1434.3, 1259.2 cmÀ1
; MS (MALDI-TOF): m/z
calcd: 854.5 [M]+; found: 775.7 [MÀBr]+.
Synthesis of compound 2: 7-Amino-4-methylcoumarin (1.75 g, 10 mmol),
bromotetradecane (22 g, 80 mmol), K2CO3 (5.6 g, 40 mmol), and NaI
(0.38 g, 2.5 mmol) were mixed in MeCN (90 mL) and the mixture was
heated at reflux for 36 h. After the reaction mixture was cooled to RT,
the solvent was evaporated in a rotary evaporator. Then, the residue was
partitioned between CH2Cl2 and brine. The organic layer was dried over
MgSO4 and concentrated. The crude product was purified by column
chromatography on silica gel (petroleum ether/EtOAc, 10:1 v/v) to
afford compound 2 (2.9 g, 52% yield). 1H NMR (CDCl3): d=7.37 (d,
1H), 6.55 (dd, 1H), 6.46 (s, 1H), 5.93 (s, 1H), 3.30 (t, 4H; NCH2), 2.33
(s, 3H; ArCH3), 1.59 (m, 4H; NCH2CH2), 1.31–1.25 (m, 44H), 0.89 ppm
(t, 6H); IR (KBr pellet): n˜ =2914.5, 2849.8, 1716.8, 1603.1, 1469.1, 1362.0,
1404.0 cmÀ1; MS (MALDI-TOF): m/z calcd: 567.5 [M]+; found: 568.5
[M+H]+.
Synthesis of 1,2-bis(2-azidoethoxy)ethane: Triethylene glycol (TEG, 1.5 g,
10 mmol) and CBr4 (7.1 g, 22 mmol) were dissolved in dry THF. PPh3
(5.3 g, 20 mmol) was added slowly under vigorous stirring. The reaction
mixture was stirred at RT for 1 h, filtered, and concentrated. The crude
material was purified by column chromatography on silica gel (petroleum
ether/EtOAc, 3:1 v/v) to afford 1,2-bis(2-bromoethoxy)ethane as a color-
less oil (2.5 g, 90% yield). 1H NMR (CDCl3): d=3.82 (t, 2H;
BrCH2CH2), 3.68 (t, 2H; BrCH2CH2), 3.46 ppm (t, 2H;
BrCH2CH2OCH2). Then, 1,2-bis(2-bromoethoxy)ethane (1.0 g, 3.6 mmol)
and sodium azide (0.5 g, 7.5 mmol) were mixed in DMF (40 mL) and stir-
red at 608C for 6 h. The mixture was filtered and the filtrate was concen-
trated in vacuum. The crude product was purified by column chromatog-
raphy on silica gel (petroleum ether/EtOAc, 4:1 v/v) to afford 1,2-bis(2-
azidoethoxy)ethane as
a
colorless oil (0.6 g, 82% yield). 1H NMR
Synthesis of compound 3: Compound 2 (5.5 g, 10 mmol) and SeO2 (1.7 g,
15 mmol) were mixed in p-xylene (20 mL) and the mixture was heated at
reflux under an argon atmosphere for 6 h with vigorous stirring. Then,
the mixture was filtered, concentrated, and dissolved in ethanol/THF
(20 mL, 1:1 v/v). After NaBH4 (0.94 g, 25 mmol) was slowly added, the
reaction was stirred for 3 h at RT. The mixture was carefully neutralized
with 1m HCl and concentrated in a rotary evaporator to remove the sol-
vent. The residue was dissolved in CH2Cl2 (50 mL), washed with brine,
and dried over MgSO4. After evaporation of the solvent, the crude prod-
uct was purified by column chromatography on silica gel (petroleum
ether/EtOAc, 2:1 v/v) to give compound 3 (2.6 g, 44% yield). 1H NMR
(CDCl3): d=7.35 (d, 1H), 6.68 (dd, 1H), 6.58 (d, 1H), 6.30 (s, 1H), 4.84
(s, 2H; ArCH2OH), 3.32 (t, 4H; NCH2), 1.9 (br s; ArCH2OH), 1.59 (m,
4H; NCH2CH2), 1.31–1.28 (m, 44H), 0.89 ppm (t, 6H); IR (KBr pellet):
n˜ =3383.5, 2932.8, 1709.2, 1608.1, 1467.6, 1377.3, 1060.3, 1060.3 cmÀ1; MS
(MALDI-TOF): m/z calcd: 583.5 [M]+; found: 584.5 [M+H]+.
(CDCl3): d=3.69 (m, 4H; CH2OCH2), 3.41 ppm (t, 2H; N3CH2); MS
(ESI): m/z calcd: 200.1 [M]+; found: 223.1 [M+Na]+.
CAC determination: The CAC of amphiphile 1 was determined by using
the fluorescent-probe technique with a hydrophobic dye, Nile Red. A
certain amount of Nile Red was added to a series of aqueous solutions of
amphiphile 1 with different concentrations. These solutions were sonicat-
ed for 30 min and then left to stand for 2 h. The solutions were examined
by fluorescence spectroscopy. The emission intensity of Nile Red at
636 nm was plotted versus the logarithm of the concentration of amphi-
phile 1, thus showing a nonlinear relationship; the CAC was determined
as the inflection point (see the Supporting Information, Figure S2a). The
CAC was also determined by concentration-dependent conductivity ex-
periments. A plot of the conductivity of solutions of amphiphile 1 with
various concentrations versus the concentration of amphiphile 1 is shown
in the Supporting Information, Figure S2b. The CAC was obtained from
the extrapolated intersection of the two linear regions in the plot, as indi-
cated by an arrow.
Synthesis of compound 4:
A solution of propargyl bromide (8 g,
67 mmol) in dry CH2Cl2 (10 mL) was added dropwise to a stirring solu-
tion of ethanolamine (2 g, 33 mmol) and K2CO3 (9.5 g, 70 mmol) in dry
CH2Cl2 (30 mL) at 08C. The mixture was stirred at RT for 3 h and
poured into an excess amount of water. The crude product was extracted
with CHCl3 and the combined extracts were dried over MgSO4 and
evaporated. The crude product was purified by column chromatography
on silica gel (CH2Cl2/EtOAc, 1:1 v/v) to give compound 4 (3.6 g, 81%
yield). 1H NMR ([D6]DMSO): d=3.64 (t, 2H; HOCH2), 3.49 (d, 4H;
Preparation of the vesicles: A solution of amphiphile 1 in CHCl3 was
placed in a vessel and dried. Distilled water was added to the vessel to a
concentration of ꢀ10À3 m. The suspension was sonicated for about 15 min
and allowed to equilibrate for 2 h, thereby affording the vesicles.
Polymerization of the vesicles: 1,2-Bis(2-azidoethoxy)ethane, CuCl2
(2.5 mol%), and sodium ascorbate (25 mol%) were added to a solution
of the vesicles. Then, the mixture was stirred at RT for 24 h to afford the
click-polymerized vesicles.
ꢀ
ꢀ
NCH2C CH), 2.76 (t, 2H; OCH2CH2N), 2.26 ppm (t, 2H; NCH2C CH);
MS (ESI): m/z calcd: 137.1 [M]+; found: 138.1 [M+H]+.
Preparation of the NR-loaded vesicles: A solution of Nile Red in CH2Cl2
(50 mL, 1 mgmLÀ1) was injected into the vesicular solution of amphiphile
1. The mixture was sonicated for 30 min to induce encapsulation and
then allowed to equilibrate for 2 h. The organic solvent and excess Nile
Red were removed by evaporation and filtration, respectively.
Synthesis of compound 5: 1,1’-Carbonyldiimidazole (CDI, 1.4 g,
11 mmol) was added to
a stirring solution of compound 4 (1.4 g,
10 mmol) in dry THF (20 mL) in an ice bath. After stirring at RT for 7 h,
compound 3 (5.9, 10 mmol) and a catalytic amount of KOH were added.
The mixture was stirred at ambient temperature for a further 4 h before
being concentrated. The residue was dissolved in CH2Cl2 (60 mL),
washed with brine, and dried over MgSO4. After removal of the solvent,
the crude product was purified by column chromatography on silica gel
(petroleum ether/EtOAc, 4:1 v/v) to give compound 5 (4.9 g, 66% yield).
1H NMR (CDCl3): d=7.28 (d, 1H), 6.54 (dd, 1H), 6.44 (s, 1H), 6.16 (s,
Encapsulation of Rh6G in the polymerized vesicles: An excess of Rh6G
was added to a solution of unpolymerized vesicles. The mixture was soni-
cated for 30 min to afford unpolymerized vesicles with entrapped Rh6G.
The polymerized vesicles that contained entrapped Rh6G were prepared
by click polymerization of the loaded unpolymerized vesicles (see
above). The polymerized vesicles were dialyzed for 24 h in the dark to
remove any free Rh6G.
ꢀ
1H), 5.26 (s, 2H; ArCH2O), 4.34 (t, 2H; OCH2), 3.51 (d, 4H; NCH2C
ꢀ
CH), 3.31 (t, 4H; NCH2), 2.93 t, 2H; OCH2CH2N), 2.26 (t, 2H; NCH2C
CH), 1.59 (m, 4H; NCH2CH2), 1.31–1.24 (m, 44H), 0.89 ppm (t, 6H); IR
(KBr pellet): n˜ =3331.4, 2932.8, 2853.5, 1740.8, 1709.2, 1607.7, 1471.4,
1036.1 cmÀ1; MS (MALDI-TOF): m/z calcd: 746.5 [M]+; found: 747.1
[M+H]+.
Acknowledgements
Synthesis of amphiphile 1: Compound 5 (2 g, 2.6 mmol) and an excess
amount of bromoethane were mixed in distilled acetone and stirred for
2 days at 508C, before evaporating to dryness. The crude product was pu-
rified by column chromatography on silica gel (CH2Cl2/MeOH, 10:1 v/v)
to afford amphiphile 1 (1.2 g, 52% yield). 1H NMR (CD3OD): d=7.55
(d, 1H), 6.54 (dd, 1H), 6.44 (s, 1H), 6.16 (s, 1H), 5.26 (s, 2H; ArCH2O),
Financial support from the National Natural Science Foundation of
China (21233011, 21103210, 21172229, and 21002109) and the 973 Pro-
gram (2010CB934500, 2013CB834700, and 2013CB834505) is gratefully
acknowledged.
ꢀ
ꢀ
4.83 (t, 2H; OCH2), 4.65 (d, 4H; NCH2C CH), 4.10 (t, 2H; NCH2C
CH), 3.86 (m, 2H; NCH2CH3), 3.71 (d, 2H; NCH2CH2O), 3.46 (t, 4H;
Chem. Eur. J. 2013, 19, 7931 – 7936
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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Received: February 9, 2013
Published online: April 15, 2013
7936
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Chem. Eur. J. 2013, 19, 7931 – 7936