Towards copper-free nanocapsules obtained by orthogonal interfacial click polymerization in miniemulsion

Article abstract of DOI:10.1039/c2cc30253e

A facile method to produce nanocapsules by copper-free interfacial click -polymerization as orthogonal reaction for the encapsulation of functional molecules is successfully performed using stable miniemulsion droplets. Difunctional azides and alkynes have been used for polymerization around the miniemulsion droplets, leading to the formation of nanocapsules. The results were compared with copper-catalyzed systems.

Full text of DOI:10.1039/c2cc30253e

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COMMUNICATION
Towards copper-free nanocapsules obtained by orthogonal interfacial
‘‘click’’ polymerization in miniemulsionw
Joerg Max Siebert, Grit Baier, Anna Musyanovych and Katharina Landfester*
Received 12th January 2012, Accepted 11th April 2012
DOI: 10.1039/c2cc30253e
A facile method to produce nanocapsules by copper-free inter-
facial ‘‘click’’-polymerization as orthogonal reaction for the
encapsulation of functional molecules is successfully performed
using stable miniemulsion droplets. Difunctional azides and alkynes
have been used for polymerization around the miniemulsion
droplets, leading to the formation of nanocapsules. The results
were compared with copper-catalyzed systems.
Recently, several groups described the reaction of the
1,3-dipolar cycloaddition of terminal electron deficient alkynes
to azides at room temperature without the use of a catalyst.14,15
This reaction enables mild conditions for biological applications16,17
and can be performed in the absence of an inert atmosphere
which is needed to protect the catalyst from being oxidized.
These facts also facilitate the use of ‘‘click’’ chemistry in
(mini)emulsion systems, where degassing and the subsequent
reduction of a copper(II) catalyst are crucial steps, which have
been adapted from the AGET-ATRP procedure for the surface
functionalization of poly(propargyl acrylate) nanoparticles.18
Here, we report a new concept for the facile and selective
formation of nanocapsules consisting of polytriazoles as shell
materials and an aqueous isotonic sodium chloride core
solution. We designed an electron deficient dialkyne in order
to produce the capsules in a nanometer size range, at room
temperature and without the use of any catalyst. As a conse-
quence, the system is less complicated, since the reaction
conditions are now straightforward.
The encapsulation of sensitive compounds bearing a variety of
functional groups, such as hydroxy, thiol, or amine, into
polymeric nanocapsules under classical polymerization condi-
tions is a great challenge. Even though it was shown recently
that the miniemulsion technique is very suitable for obtaining
polymeric nanocapsules1 by either radical polymerization2 or
the polyaddition of diisocyanates and diols (or diamines)
performed at the droplets interface,3,4 the presence of radicals
can lead to e.g. oxidation and abstraction reactions,5 and
diisocyanates react easily with a wide range of nucleophiles
such as amines, alcohols, thiols and acids. Furthermore, in both
polymerization reaction types, it is difficult to avoid the interaction
of the hydrophilic ‘‘payload’’ with the monomers and the
subsequent entrapment in the polymeric chain. Therefore, for
the encapsulation of many biomolecules and other functionalized
molecules, an orthogonal reaction is required.
The polytriazoles are obtained by CuAAC polymerization
at the droplet’s interface at room temperature from a water
soluble azide and an oil soluble diyne (see Scheme 1) for
comparison with the copper free system. The used monomer
contains hydroxyl groups to enhance its solubility in water.
Furthermore, after the reaction the residual hydroxyl groups
can be used for the post functionalization of the capsule’s
surface. Due to the mild reaction conditions used for the
formation of nanocapsules, we believe that the reported
method has a high potential for the (nano)encapsulation of
sensitive compounds at high yields.
‘‘Click’’ and especially the copper catalyzed azide–alkyne
1,3-dipolar cycloaddition (CuAAC) is a highly atom efficient
reaction, since no side products are produced and a very stable
aromatic triazole ring is formed.6–8 The ligands for copper
catalysts are readily available and both reaction components
(i.e. alkyne and an azide) could be easily synthesized or
supplied from commercial sources. All these properties make
the ‘‘click’’ reaction attractive for a wide range of applications
in materials science for surface functionalization9–11 and the
synthesis of functionalized polymers11,12 as well as for the
facile synthesis of dendrimers.13 Moreover, the CuAAC reac-
tion is ultimately bio-orthogonal, as neither azide nor
terminal alkyne functional groups are generally present in
the nature.
The diazide monomer 2,2-bis(azidomethylene)-1,3-propanediol
(BAP) was synthesized according to the one-step procedure
reported by Sharpless et al.7 with 95% yield and good water
Max-Planck-Institute for Polymer Research, Ackermannweg 10,
D-55128 Mainz, Germany. E-mail: landfester@mpip-mainz.mpg.de;
Fax: +49 6131 379100; Tel: +49 6131 379130
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc30253e
Scheme 1 Schematic representation of a step growth ‘‘click’’ poly-
merization reaction at the droplet’s interface and the chemical structure
of the used monomers.
c
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solubility up to 25 wt%. In a typical capsule synthesis
(for detailed information, see ESIw), BAP was dissolved in
1 mL of 0.9% NaCl solution together with the catalyst precursor
[Cu(II)(BA6TREN)]Br2. For visualization as well as release
experiments, the hydrophilic fluorescent dye sulforhodamine
SR101 was encapsulated. As continuous phase, cyclohexane
was used, in which the organo-soluble alkynes were dissolved
together with the surfactant poly((ethylene-co-butylene)-b-(ethylene
oxide)), P(E–B-b-EO).19 The reaction was started after addition of
organo-soluble tin(II)-2-ethylhexanoate to reduce copper(II) to
copper(I). The reaction was performed at room temperature as
well as at 50 1C. It turned out that the reaction performed at
50 1C was completed after 24 h, whereas 48 h were required for
the reaction carried out at room temperature as monitored
by SEC and IR-spectroscopy. The polymerization using
hexanediol dipropiolate (HDDP) was carried out under the
same conditions as mentioned above but without use of any
catalyst, reducing agent and inert atmosphere. The obtained
yields in both cases were quantitative.
(in the case of OD and HDDP) is slightly smaller and the size
distribution is broader as compared to the initial miniemulsion
droplets (250 nm with a standard deviation (SD) of 18%). The
obtained results could be assigned to the droplets disruption
caused by argon bubbling through the miniemulsion system
which is necessary for performing the click reaction. Capsules
with a broad size distribution around 214 nm (SD, 43%) were
found when DEB was used as an alkyne monomer, which
could be due to its poor solubility in the continuous phase.
By using an electron deficient propiolic acid diester
(HDDP), it was possible to perform the reaction at room
temperature without the presence of any catalyst, reducing
agents or inert atmosphere. The degree of polymerization
(DP), determined by SEC, was in a range between 41 (number
corresponds only to the dissolved/analyzed part of the polymer)
and 97 or above for both catalyzed (OD/DEB) and non-catalyzed
(HDDP) systems. Moreover, the DPs for all miniemulsion
systems were found to be very close to each other, indicating
that the reaction was completed and the molecular weight was
limited by impurities. The monomers conversion was also followed
The chemical composition of the polymer formed at the
droplets interface (heterophase) was compared with a reference
polymer obtained using ‘‘click’’ polymerizations in a single
phase DMF solution. NMR studies of both polymers showed
no difference in the chemical composition. The obtained data
are summarized in Tables 1 and 2. The molecular weights of the
polymer synthesized in the miniemulsion were lower compared
to the ones obtained in solution. When using DEB as dialkyne,
polymers became insoluble and it was necessary to apply heat in
order to dissolve them in DMF for the analysis. The polymers’
molecular weights possessed a typical polydispersity of around
two, as expected for a step growth polymerization. Reactions were
carried out using 1,7-octadiyne (OD) and 1,4-diethynylbenzene
(DEB) as a commercially available dialkyne source. Already
the first produced nanocapsules showed a size range of 200 nm
as determined by dynamic light scattering (DLS) and verified
by electron microscopy. The average size of the capsules
by IR spectroscopy. The vibration band at B2100 cmÀ1
,
which is characteristic of the azide and alkyne, almost
disappeared, thus indicating that the reaction is completed
(for spectra see Fig. S1, ESIw).
While DEB, when used as an alkyne source, yielded hardly
soluble polymers, polymers with OD showed moderate and
HDDP showed good solubility in DMF. Thermal analysis of
the polymers synthesized in a solution (Table 2) revealed a
thermal stability up to 360 1C. DSC data show that the
obtained polymers are semi-crystalline.
To monitor the reaction, the kinetics of HDDP and BAP
polymerization was studied in a NMR tube using DMSO as a
solvent. The NMR spectra were periodically recorded within
48 h (Fig. 1). NMR spectroscopy revealed a mixture of 1,4-
and 1,5-isomers, detected by two signals at 8.58 and 8.63 ppm
of the characteristic sp2-C–H proton of the formed triazole
double bond. The characteristic signal from the monomers’
methylene groups at 3.29 ppm decreased over time with new
broad signals appearing between 3.17 and 3.25 ppm when the
polymerization proceeded. An exponential decay for the rela-
tions of the signal at 3.29 ppm and the two signals at around
8.60 ppm was found.
Table 1 Characterization of the obtained polymers from miniemulsion
systems
Da
Mn/Mw
Db/nm
Monomer
Mwa/g molÀ1
DP
(SDc (%))
HDDP (without Cu)
OD (with Cu)
DEB (with Cu)
33 600
22 480
24 590d
2.25
2.23
2.35
82
78
81
212 (28)
232 (24)
214 (43)
All dispersions show a good shelf life stability of several
weeks. The morphology of the capsules was studied by electron
microscopy (Fig. 2). The shell of the capsules obtained from
DEB and HDDP (in SEM images) looks to be more stable and
a
b
Determined by SEC, DMF as eluent, 60 1C. As determined by
c
d
DLS. SD = standard deviation, determined by DLS. Polymer was
not completely soluble.
Table 2 Characterization of the obtained polymers from solvent
systems
Da
Monomer Mwa/g molÀ1 Mn/Mw DP Tdecomc/1C Tgd/1C Tmd/1C
HDDP
OD
DEB
45 500
28 400
12 400b
2.17
1.75
2.13
101 240
97 350
41 360
67
84
—
142
155
163
a
b
Determined by SEC, DMF as eluent, 60 1C. Polymer was not
completely soluble. Determined by TGA, using the onset point.
Fig. 1 Left: kinetics of HDDP and BAP polymerization followed by
NMR spectroscopy. Right: product to starting material ratio followed
over time and determined from NMR data.
c
d
Determined by DSC.
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slightly lower as compared to the aqueous core and the leakage
of the fluorescent dye was slightly higher (around 9%). The
obtained results indicate that the capsule’s polymeric shell
possesses high compactness and resistance against leakage over
time. The initial amount of free dye (between 2% and 6%)
could be due to the centrifugation process leading to the
destruction of nanocapsules.
The biocompatibility of the capsules was studied by an MTS
test. The results revealed no cytotoxicity within a 24 h incubation
time. The amount of dead cells was comparable with the negative
control (o0.5%). Therefore, the concentration of possibly
remaining copper ions or tin compounds is low enough to be
tolerated by the cells, thus making these materials biocompatible.
In summary, we report for the first time the use of ‘‘click’’
reactions to form nanocapsules through polymerization of low
molecular weight difunctional monomers at the interface of
miniemulsion droplets. The concept of interfacial step growth
addition of electron deficient alkynes and a water soluble azide
showed that there is no need of a catalyst. The reported system
was compared to a poly-CuAAC in the presence of a surface
active catalyst. In both cases, high molecular weight polymers
were achieved within 48 h under mild conditions. The
proposed systems are of high interest for biological systems,
where the amount of catalysts and other additional reagents
should be kept as low as possible. All polymerizations could be
conducted at room temperature, resulting in a high degree of
polymerization. The obtained polymeric shells are compact
with a low rate of hydrophilic ‘‘payload’’ leakage, as demon-
strated by diffusion experiments on the example of hydrophilic
fluorescent dye SR101.
Fig. 2 Upper row: SEM images of nanocapsules; middle row: TEM
images obtained by drop casted dispersion of capsules; bottom row: TEM
images of EPON embedded and ultrathinly sectioned capsule samples. All
samples used for TEM analysis were stained with OsO4 to improve the
contrast of the polymers (OD: octadiyne; DEB: diethynylbenzene;
HDDP: hexanediol dipropiolate); core: aqueous 0.9 NaCl solution.
does not collapse under the high vacuum conditions. The shell
thickness was determined from the TEM images (around 14 nm
for DEB and HDDP and around 10 nm for OD). Using OD led
to smaller wall thicknesses which can be seen in the SEM
images (collapsed nanocapsules, upper row: second image in
Fig. 2). For a better visualization of the polymeric shells, the
capsules were embedded in EPON resin (bottom row, Fig. 2;
for experimental details see ESIw) and sectioned using an
ultramicrotome. In all cases the ‘‘core–shell’’ morphology
was found. For sectioned samples a deformation of the
capsules could not be avoided, but for drop casted samples,
slightly deformed spherical capsule structures could be seen.
After the synthesis, all nanocapsules were transferred into the
aqueous phase by redispersing them in water containing the
anionic surfactant SDS. The size distributions of the nano-
capsules did not change, indicating that no aggregation or
swelling of capsules had taken place. Also the electron micro-
scopy analysis of both cyclohexane and waterborne dispersions
showed the same morphology.
Notes and references
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The influence of the monomer type on the shell permeability
was studied on capsules with the fluorescent dye SR101 as a
‘‘payload’’. The release kinetics was monitored by fluorescence
spectroscopy for a period of 40 days. For this purpose, the
redispersed non-dialyzed samples were centrifuged at 6000 rpm
for 30 min and the supernatant was analyzed in a plate reader,
to determine the fluorescence intensity (for details see ESIw,
Fig. S2).
The obtained data (Fig. S3 in ESIw) revealed that only a
small fraction of the dye (around 7% related to the introduced
amount) was released during the observed period of time,
indicating a good impermeability of the polymer shells. For
encapsulation of more hydrophobic, e.g., biological substances,
DMSO as a solvent is generally used. Therefore, we investigated
the formation of nanocapsules using DMSO as dispersed phase.
The capsules in the size range of 280 nm were obtained for all
dialkynes. The molecular weights of the resulting polymers were
14 J. A. Codelli, J. M. Baskin, N. J. Agard and C. R. Bertozzi, J. Am.
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5472 Chem. Commun., 2012, 48, 5470–5472
This journal is The Royal Society of Chemistry 2012