Surface click reactions on polymeric nanocapsules for versatile functionalization

Article abstract of DOI:10.1021/ma300312n

One-step synthesis of poly(butyl cyanoacrylate-co-propargyl cyanoacrylate) (P(BCA/PCA)) and polyurethane (PUR) azide nanocapsules for direct covalent and variable functionalization is reported. The capsules in the size range between 230 and 350 nm were sy

Full text of DOI:10.1021/ma300312n

Article
pubs.acs.org/Macromolecules
Surface Click Reactions on Polymeric Nanocapsules for Versatile
Functionalization
Grit Baier, Joerg Max Siebert, Katharina Landfester, and Anna Musyanovych*
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
ABSTRACT: One-step synthesis of poly(butyl cyanoacrylate-co-propargyl cyanoa-
crylate) (P(BCA/PCA)) and polyurethane (PUR) azide nanocapsules for direct
covalent and variable functionalization is reported. The capsules in the size range
between 230 and 350 nm were synthesized in an inverse (water-in-oil)
miniemulsion system through either interfacial anionic polymerization (in the
case of P(BCA/PCA)) or interfacial polyaddition reaction with 2,4-toluene
diisocyanate (PUR azide). Surface functionalization of nanocapsules was achieved
by different copper-catalyzed azide−alkyne cycloaddition (CuAAC) reactions,
which were carried out under mild and ambient conditions. Click reactions of
rhodamine azide and anthracene azide to P(BCA-co-PCA) nanocapsules were
performed for quantitative determination of the surface alkyne bonds. PUR-azide
nanocapsules were functionalized with propiolic acid and fluorescent alkyne dye.
The reaction kinetics between propiolic acid and the azide monomer was studied by
1H NMR spectroscopy. Furthermore, the functionalization of nanocapsules with
propiolic acid was extended to copper-free azide−alkyne cycloaddition, thus making the system suitable for copper-sensitive
materials.
reaction conditions and results in high yields.20,21 To date,
numerous click-chemistry-based strategies have been reported
INTRODUCTION
In the past decades there is a growing interest in the
■
for the modification of surfaces. Over the past few years, several
development of nanocarriers with well-defined structures and
click reactions have been particularly elaborated for the
tailorable properties for drug delivery systems. The particle size,
shape, and surface functionality are the most important factors
that influence the distribution of the particles within the body,
modification of curved surfaces, i.e., micro- and nanoparticles.
Ranjan and Brittain22 combined living radical polymerization
their interaction, and uptake by living cells.1−4 Colloidal carriers
with click chemistry to modify the surface of silica nanoparticles
with polymers. The possibilities of postfunctionalization of
such as liposomes, micelles, dendritic polymers, and nano-
poly(6-azidohexyl methacrylate)-grafted silica nanoparticles
particles or nanocapsules are the most promising candidates
with various functional alkynes via click reactions were also
with regards to the site-specific targeting and controlled drug
demonstrated by Li and Benicewicz.23 Using Cu-catalyzed click
reaction, the modification of polymeric particles with azide- or
alkyne-functionalized dyes and macromolecules, i.e., poly-
(ethylene oxide), was successfully achieved in aqueous and
nonaqueous environments.24−26 Ouadahi et al.27 reported the
synthesis of azide-functionalized polystyrene nanoparticles,
which were used as clickable polymeric scaffolds for the
grafting of dansyl and fluorescein derivatives through copper-
(I)-catalyzed azide−alkyne cycloaddition. For localized cell
delivery, self-assembled polymeric nanoparticles containing
azide groups were modified with targeting alkyne-functionalized
ligands by the aqueous Huisgen’s 1,3 dipolar cycloaddition
reaction, catalyzed by copper sulfate and sodium ascorbate.28
Magnetic poly(N-propargylacrylamide) microspheres were
used as the anchor to enable a “click” reaction with an azido-
end-functionalized model peptide.29 The versatility of copper-
release.5−8 The main advantages to use nanocapsules for drug
delivery are the efficient protection of a drug against
degradation caused by the influence of the environment, and
preventing desorption of a drug, as it could be the case with
physically adsorbed drug molecules onto the particle surface.
In recent years, the fabrication of polymer nanocapsules
made from different materials and using various approaches was
described by many research groups ranging from the layer-by-
layer approach of preformed polymer(s) to the interfacial
reactions of monomers at stable nanodroplets.9−13 For the drug
delivery application it is of immense interest that the capsules
are biocompatible and possess functional groups on the surface
that enable covalent attachment of biomolecules for the site-
specific targeting. The covalent attachment can be achieved by
numerous coupling reactions, such as amine/epoxide, amine/
glutaraldehyde, thiol/maleimide, carbodiimide, and so on.14−16
Click chemistry, which is based on the copper-catalyzed azide−
alkyne cycloaddition, reported simultaneously in 2001/2002 by
Sharpless et al.17,18 and Meldal et al.,19 is a very efficient
strategy for the functionalization as it proceeds at the mild
Received: February 15, 2012
Revised: March 30, 2012
Published: April 11, 2012
© 2012 American Chemical Society
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Scheme 1. Schematical Presentation of the Alkyne-Functionalized PBCA/P(BCA-co-PCA) and Azide-Functionalized
Polyurethane Nanocapsules
catalyzed alkyne−azide coupling (CuAAC) in functionalizing
drug-loaded polymer nanoparticles was demonstrated via the
modification of surface of acetylene-functionalized nanoprticles
with folate, biotin, and gold nanoparticles30 Using click
chemistry, the surface functionalization of poly(N-vinylpyrro-
lidone) capsules with antibodies that are specific to colorectal
cancer cells was recently published by Caruso et al.31
either interfacial anionic polymerization (P(BCA/PCA)) or
interfacial polyaddition reaction (PUR azide). The presence of
propargyl and azide groups on the surface of P(BCA/PCA) and
PUR nanocapsules, respectively, enables to achieve high yields
of functionalization using the click chemistry. Different
fluorescent markers were attached onto the surface of
P(BCA/PCA) and PUR nanocapsules through Cu(I)-catalyzed
azide−alkyne cycloaddition (CuAAC) reactions performed
under mild and ambient conditions (Scheme 1). Furthermore,
in order to avoid any cytotoxic and side effects related to the
copper,39 the concept was extended to the copper-free azide
alkyne cycloaddition. In this way, PUR capsules functionalized
with any functional group can be obtained; as an example we
show here the functionalization with carboxylic groups by using
propiolic acid. We believe that the approach disclosed in this
paper can be applied to prepare a wide range of hybrid
polycyanoacrylate- and polyurethane-based capsules with
advanced properties.
Poly(alkyl cyanoacrylates) (PACAs) have gained an
increased clinical interest mainly due to their biodegradability,
low toxicity, and enhancement of the drug intracellular
penetrations. In the mid-1960s, cyanoacrylates were widely
employed as bioadhesives in surgery and later in the 1980s as
the polymeric carriers for delivery of biomolecules.32−37
However, the attachment of specific target molecules onto
the surface of PACAs nanocarriers still remains a big challenge.
The most commonly used systems for preparing of the drug-
loaded (PACA) particles/capsules is the anionic polymerization
initiated by a nucleophilic group. Depending on the used
surfactant (or cosurfactant), a certain amount of functional
groups might remain on the surface of nanocarriers after
polymerization. Recently, Nicolas et al.38 described the
preparation of functionalized PACA-based nanoparticles made
from a preformed poly(hexadecyl cyanoacrylate)-co-azidopoly-
(ethylene glycol) cyanoacrylate copolymer. The obtained
nanoparticles display azide functionalities at the extremity of
the PEG chains, which were further reacted with the model
alkynes by click chemistry.
EXPERIMENTAL SECTION
■
Materials. All chemicals were used as received without further
purification. Demineralized water was used throughout the experi-
ments. The monomers n-butyl cyanoacrylate and propargyl
cyanoacrylate (1H NMR (CDCl3, 400 MHz, δ in ppm): 2.59 (t, J =
2.5 Hz, alkyne), 4.88 (t, J = 2.5 Hz, 2H, =CH2), 6.72 (s, 1H, vinyl),
7.13 (s, 1H, vinyl) were kindly donated by Henkel AG & KGaA,
Duesseldorf, Germany. The oil Miglyol 812N (caprylic/capric
triglycerides) was a donation from Sasol, Germany. 2,4-Toluene
diisocyanate (TDI, 99%) and cyclohexane (>99.9%) were purchased
from Sigma-Aldrich. The oil-soluble block copolymer surfactant
poly[(ethylene-co-butylene)-b-(ethylene oxide)], P(E/B-b-EO), con-
sisting of a poly(ethylene-co-butylene) block (Mw = 3700 g mol−1) and
a poly(ethylene oxide) block (Mw = 3600 g mol−1) was synthesized
In this work we describe the one-step synthesis of poly(butyl
cyanoacrylate-co-propargyl cyanoacrylate) (P(BCA/PCA)) and
polyurethane (PUR) azide nanocapsules for direct covalent and
variable functionalization. Both types of capsules were
synthesized in inverse (water-in-oil) miniemulsion through
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starting from ω-hydroxypoly(ethylene-co-butylene), which was dis-
solved in toluene, by adding ethylene oxide under the typical
conditions of an anionic polymerization.40 P(E/B-b-EO) was
vacuum-dried prior to use. The anionic surfactant sodium dodecyl
sulfate (SDS, 99%) was purchased from Fluka. The nonionic block
copolymer Lutensol AT50 (BASF), which is a poly(ethylene oxide)
hexadecyl ether with an EO block length of about 50 units, was used as
surfactant. The surfactants Span80 and Tween80 were supplied by
Aldrich. Ethanol (Aldrich), dimethylformamide (Aldrich), ethyl
acetate (VWR), methylene chloride (<50 ppm water content)
(Acros Organics), methanol (Fisher Scientific), dichloromethane
(VWR), and chloroform (VWR) were of HPLC grade. Alexa
Fluor488 alkyne dye was purchased from Invitrogen molecular probes,
and Rhodamine B (90%) was purchased from Aldrich. Sodium azide
(98%) was purchased from SERVA. 9-Chloromethylanthracene (97%)
was purchased from Riedel de Haen. Dicyclohexylcarbodiimide
(DCC), sodium hydrogen carbonate, (dimethylamino)pyridine,
ascorbic acid, magnesium sulfate, and sodium chloride were purchased
from Aldrich. 1-Bromo-3-propanol (97%), propiolic acid (95%), and
2,2-bis(bromomethyl)-1,3-propanediol were purchased from Acros
Organics. PBS buffer was purchased from Invitrogen. The used copper
complex Cu(II)Br2/PMDETA was synthesized using a literature
procedure.41
(MeOH) mixture (9:1). 4.6 g of a dark violet solid was obtained
after removal of the solvent (79%, 8.7 mmol). Rf (DCM/MeOH, 9:1):
0.36. 1H NMR (CDCl3, 300 MHz, δ in ppm): 8.25 (1H, dd, 4J = 1.37
Hz, J = 7.8 Hz), 7.82−7.69 (m, 4H), 7.28 (dd, 1H, J = 1.3 Hz, J =
7.5 Hz), 7.04 (d, 2H, 3J = 9.0 Hz), 6.88 (dd, 2H, 4J = 2.4 Hz, 3J = 9.2
3
4
3
3
3
Hz), 6.78 (d, 2H, J = 2.4 Hz), 4.08 (m, 2H), 3.61 (q, 8H, J = 7.1
3
3
Hz), 3.17 (t, 2H, J = 6.7 Hz), 1.71 (m, 2H), 1.29 (t, 12H, J = 7.1
Hz). 13C NMR (CDCl3, 75 MHz, δ in ppm): 165.1, 158.8, 157.9,
155.8, 133.7, 133.4, 131.5, 131.4, 130.7, 130.5, 129.9, 114.5, 113.7,
96.6, 76.8, 63.9, 62.7, 48.9, 48.2, 46.3, 29.4, 28.1, 12.8. ESI-MS: 526
(100, M+), 527 (25, [M + H]+).
Synthesis of 9-Methylenazidoanthracene (Anth-N3). The
synthesis was performed following a slightly modified procedure
Scheme 4. Synthesis of Anthracene-N3 (Anth-N3)
described by Xie et al.43 (Scheme 4). Briefly, 2.26 g of 9-
chloromethylanthracene (10 mmol) was dissolved in 50 mL of
dimethylformamide (DMF), and 1.95 g of sodium azide (30 mmol)
was added. The solution was heated for 24 h to 50 °C using an oil
bath. After the reaction was finished, DMF was evaporated using a
rotoevaporator, and the resulting solid was dissolved in 100 mL of
chloroform and washed twice with 150 mL of water. The organic
phase was dried over magnesium sulfate and filtered, and the solvent
was evaporated. The resulting solid was recrystallized from ethanol,
and the solution was cooled to 0 °C using an ice bath, where it
crystallized to yellow needlelike structures. After drying the substance
in vacuum, 1.5 g of yellow solid was obtained (64%). 1H NMR
Synthesis of 1-Azido-3-propanol. 25 g of 1-bromo-3-propanol
(180 mmol, 1.0 equiv) was dissolved in 100 mL of dimethylforma-
mide, and 23.4 g of sodium azide (353 mmol, 2.0 equiv) was added.
The mixture was heated to 50 °C for 24 h using an oil bath. The
formed precipitate was filtered out, and DMF was removed at 60 °C
using a rotoevaporator. The resulting liquid was dissolved in 250 mL
of ethyl acetate and washed with 200 mL of water and brine each. The
organic phase was dried using magnesium sulfate and filtered, and
ethyl acetate was distilled out, using a rotoevaporator. 10.1 g (56%,
100 mmol) of a colorless liquid were obtained. 1H NMR (CDCl3, 300
3
MHz, δ in ppm): 3.70 (m, 2H), 3.41 (t, 2H, J = 3.41 Hz), 1.79 (m,
2H). 13C NMR (CDCl3, 75 MHz, δ in ppm): 60.0, 48.6, 31.6.
3
(CDCl3, 300 MHz, δ in ppm): 8.49 (s, 1H), 8.28 (dd, 2H, J = 9.0
3
Hz), 8.03 (d, 2H, J = 9.0 Hz), 7.58 (m, 2H), 7.50 (m, 2H), 5.31 (s,
Scheme 2. Synthesis of 1-Azido-3-propanol
2H). 13C NMR (CDCl3, 75 MHz, δ in ppm): 131.6, 130.9, 129.5,
129.2, 127.1, 125.9, 125.4, 123.7, 46.6. IR (ν in cm−1): 3446 (b), 2096
(s), 2055 (s), 1228 (s), 858 (s), 738 (s). FD-MS: 233.3 M+(100),
234.4 (M + H)+ (22).
Synthesis of 2,2-Bis(azidomethyl)-1,3-propanediol (BAP).
Synthesis of 1-Azido-3-propylrhodamine Ester (Rho-N3). The
synthesis was performed using a modified literature procedure42
(Scheme 3). 5.3 g of Rhodamine B (11 mmol, 1.0 equiv) was dissolved
in 100 mL of dry dichloromethane under an argon atmosphere in a
flame-dried Schlenk vessel. 1.2 g of 1-azido-3-propanol (12 mmol, 1.1
equiv) and 12 mg of (dimethylamino)pyridine (0.1 mmol, 0.01 equiv)
were added, and the solution was cooled to 0 °C in an ice bath. 4.12 g
of dicyclohexylcarbodiimide was added, and the solution was stirred
and warmed to room temperature over 12 h. The formed white
precipitate was filtered out, and 100 mL of dichloromethane (DCM)
was added. The organic solution was washed with 2 × 200 mL of
saturated NaHCO3 solution and 200 mL of water. The organic phase
was dried over magnesium sulfate and filtered, and the solvent was
evaporated using a rotoevaporator. The resulting dark violet solid was
submitted to column chromatography, using a DCM/methanol
BAP was synthesized using a modified literature procedure44 (Scheme
Scheme 5. Synthesis of 2,2-Bis(azidomethyl)-1,3-
propanediol (BAP)
5). 15 g of 2,2-bis(bromomethyl)-1,3-propanediol (57.3 mmol) was
dissolved in 200 mL of dimethylformamide, and 15 g of sodium azide
Scheme 3. Synthesis of 1-Azido-3-propylrhodamine Ester (Rho-N3)
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was dispersed in the solution. The mixture was heated for 24 h at 120
°C and formed sodium bromide after cooling. The excess sodium
azide was filtered out. DMF was evaporated using a rotoevaporator,
and the resulting liquid was dissolved in 200 mL of ethyl acetate. The
organic phase was washed twice with 200 mL of water and dried over
magnesium sulfate. After filtration the solvent was evaporated by a
rotoevaporator. After drying in an oil pump vacuum for 2 days, the
obtained colorless oil solidified into white crystals upon standing. 10.1
g of a white solid were obtained (95%). 1H NMR (CDCl3, 300 MHz, δ
g of the nanocapsules was redispersed either in 10.0 g of SDS solution
(0.3 wt %) or in 10.0 g of Lutensol AT50 solution (0.3 wt %). Then,
the samples were redispersed for 20 min in a sonication bath (power
50%, 25 kHz) and 24 h dialyzed (MWCO 12 000 g mol−1) against
demineralized water in order to remove residues of the surfactant.
Afterward, the obtained azide-functionalized polyurethane nano-
capsules were subjected to the click reaction experiments.
Click Reaction of Rho-N3 and Anth-N3 to P(BCA-co-PCA)
Nanocapsules. The click reaction between either the azide end
group-functionalized rhodamine (Rho-N3) or anthracene (Anth-N3)
and P(BCA-co-PCA) nanocapsules was performed in the following
way: For the Rho-N3 click reaction, either PBCA or P(BCA-co-PCA)
nanocapsules (500 μL, solid content 1 wt %) were redispersed in a
Rho-N3 (12.4 mg, 0.0235 mmol), ethanol, Tween80 solution (0.6 wt
%). The obtained dispersion and the Cu(II)Br2/PMDETA (0.94 mg,
0.0024 mmol) were mixed together and put into an argon-flushed
Schlenk tube. The dispersion was degassed by argon bubbling for 15
min. The click reaction was initiated by injection of 1 equiv (100 μL)
of ascorbic acid (0.03 M in water) into the nanocapsules dispersion
under an argon atmosphere. The mixture was stirred for 24 h at 23 °C.
After the clicking procedure the nanocapsules were cleaned four times
by repetitive centrifugation/redispersion in ethanol in order to remove
the residuals of the clicking reagents and the nonreacted Rho-N3. The
efficiency of click reaction was determined by fluorescence spectros-
copy, measuring the fluorescence intensity of the PBCA and P(BCA-
co-PCA) freeze-dried and dissolved in THF nanocapsules.
For the click reaction with Anth-N3, 5.5 mg (0.0235 mmol) of
Anth-N3 was used, and the reaction with PBCA and P(BCA-co-PCA)
nanocapsules was performed in the same manner as described above.
Clicking of Alkyne Dye to Azide-Functionalized Polyur-
ethane Nanocapsules. For the alkyne dye click reaction, 2 mL
(solid content 1 wt %) of the azide-functionalized polyurethane
nanocapsules redispersed in Lutensol AT50, the alkyne dye (0.5 mg,
6.46 × 10−4 mmol), and Cu(II)Br2/PMDETA (0.94 mg, 0.0024
mmol) were put into an argon-flushed Schlenk tube and degassed by
argon bubbling through the dispersion for 15 min. The click reaction
was initiated by injection of 1 equiv (100 μL) of ascorbic acid (0.03 M
in water) into the nanocapsules dispersion under an argon
atmosphere. The mixture was stirred for 48 h at 25 °C. After the
click reaction the nanocapsules were cleaned four times by repetitive
centrifugation in order to remove the residuals of the reagents. The
supernatant was analyzed for the presence of nonbonded residual dye.
Cu-Free Reaction between Propiolic Acid and Azide-
Functionalized Polyurethane Nanocapsules. For the propiolic
acid click reaction, 111.5 mg (100 μL, 0.166 mmol) of propiolic acid
was added to 5.0 mL (solid content 1 wt %) of azide-functionalized
polyurethane nanocapsules redispersed in SDS solution. The mixture
was stirred for 48 h at 25 °C. After the click reaction the nanocapsules
were dialyzed (MWCO 12 000 g mol−1) against demineralized water
for 2 days in order to remove any residuals of the nonreacted propiolic
acid.
Investigation of Cu-Free Reaction Kinetics between
Propiolic Acid and Azide Monomer in Solution. 100 mg of
2,2-bis(azidomethyl)-1,3-propanediol (0.054 mmol, 1.0 equiv) was
dissolved in 0.7 mL of d6-DMSO and filled into a NMR tube. The
reaction was started by adding 72.5 mg of propiolic acid (65 μL, 0.108
mmol, 2.0 equiv). After defined periods of time the NMR spectra were
recorded.
Characterization. The average size and the size distribution of the
nanocapsules were measured by means of dynamic light scattering
(DLS) at 20 °C using a PSS Nicomp Particle Sizer 380 (Nicomp
Particle Sizing Systems) equipped with a detector for a scattering angle
of 90°. The zeta potential of the nanocapsules was measured in 10−3 M
potassium chloride solution with a Zeta Nanosizer (Malvern
Instruments, U.K.) at 20 °C. Scanning electron microscopy (SEM)
images were recorded by using a field emission microscope (LEO
(Zeiss) 1530 Gemini, Oberkochen, Germany) working at an
acceleration voltage of 170 V. The samples were prepared by diluting
the capsule dispersion in cyclohexane or demineralized water (in case
of the redispersed samples) to about of 0.01% solid content. Then, one
3
in ppm): 4.74 (t, 2H, J = 4.9 Hz), 3.28 (m, 8H). 13C NMR (CDCl3,
75 MHz, δ in ppm): 59.8, 51.1, 45.5.
Synthesis of PBCA/P(BCA-co-PCA) Nanocapsules. The PBCA/
P(BCA-co-PCA) nanocapsules were prepared through anionic
polymerization at the inverse (water-in-oil) miniemulsion droplets
interface using the previously published procedure with slight
changes.37 The used amounts of both monomers, namely n-butyl
cyanoacrylate (BCA) and propargyl cyanoacrylate (PCA), are shown
Table 1. Characterization Data for the Poly(BCA-co-PCA)
Capsules
BCA/PCA,
mmol
PBS
buffer, g
diam, nm/
(std dev, %)
mol wt (Mw),
sample
g/mol
GB-cc-1
GB-cc-2
GB-cc-3
GB-cc-4
GB-cc-5
GB-cc-6
GB-cc-7
GB-cc-8
0.94/−
1
1
1
1
2
2
2
2
305/(28)
280/(24)
235/(33)
230/(32)
310/(27)
350/(30)
340/(27)
340/(28)
37 218
34 789
19 547
23 049
98 146
54 916
37 690
30 408
0.70/0.24
0.47/0.47
0.24/0.70
0.94/−
0.70/0.24
0.47/0.47
0.24/0.70
in Table 1. The miniemulsions were stabilized with a mixture of
surfactants (Span80 and Tween80). The continuous phase (7.6 g)
consisted of 0.152 g of Span80, 0.228 g of Tween80, and Miglyol
812N as oil. Different amounts of PBS buffer (see Table 1) were used
as an aqueous phase. The aqueous phase was slowly added to the oil
phase under stirring and ice cooling. Thereafter, stirring was continued
for half an hour at 4 °C. Afterward, the samples were homogenized
using a Branson 450 W sonifier with an inverse cup tip (60 s, 5 s
sonication, 10 s pause, 50% amplitude) at 4 °C. Shortly after the
homogenization, an earlier prepared mixture of BCA/chloroform or
BCA/PCA/chloroform was added dropwise to the miniemulsion
under gentle stirring, and the formation of polymer around the
aqueous droplets took place over 4 h at 25 °C. The monomer(s) to
chloroform volume ratio corresponds to 100 μL/300 μL, and it was
kept constant for all experiments. After polymerization the obtained
nanocapsules were separated from the oil phase by centrifugation
(Sigma, 3k-30, RCF 17968, 20 min), washed in ethanol, and used for
click reaction experiments.
Synthesis of Azide-Functionalized Polyurethane Nano-
capsules. The azide-functionalized polyurethane nanocapsules were
prepared through polyaddition reaction at the inverse (water-in-oil)
miniemulsion droplets interface using the previously published
procedure.13,45 The formulation process is shown in Scheme 6.
Initially, 100 mg of 2,2-bis(azidomethyl)-1,3-propanediol was mixed
with 30 mg of sodium chloride and 1.3 g of demineralized water. Then,
100 mg of P(E/B-b-EO) was dissolved in 7.5 g of cyclohexane at 50
°C using an ultrasonication for 180 s with a 90% amplitude in a pulse
regime (20 s sonication, 10 s pause) bath. Both solutions were mixed
together, stirred over 1 h at 25 °C, and ultrasonicated using a Branson
W450-Digital sonifier (1/2 in. tip) under ice cooling in order to
prevent evaporation of the solvent. A solution consisting of 5 g of
cyclohexane, 30 mg of P(E/B-b-EO), and 100 of mg TDI was added
dropwise over 5 min to the earlier prepared mixture. The reaction was
carried out overnight at 25 °C. After synthesis the nanocapsules were
washed twice with cyclohexane. The azide-functionalized polyurethane
nanocapsules were centrifuged, the supernatant was removed, and 2.0
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Scheme 6. Synthesis of Azide-Functionalized Polyurethane Nanocapsules and Their Subsequent Redispersion in the Aqueous
Phase
Figure 1. (A, B) SEM images of redispersed PBCA (sample GB-cc-5) and P(BCA-co-PCA) (sampleGB-cc-8) nanocapsules, respectively. (C) TEM
image of P(BCA-co-PCA) (sampleGB-cc-7) nanocapsules. Dark circles correspond to the polymeric shell.
droplet of the sample was placed onto a silica wafer and dried under
ambient conditions overnight. The solid content of the capsule
dispersion was measured gravimetrically. The analysis of the polymer
was performed by FTIR measurements. The sample powder was
obtained by freeze-drying the capsule dispersion for 48 h at −60 °C
under reduced pressure. Then 3 mg of the dry sample was pressed
with KBr to form a pellet, and a spectrum between 4000 and 400 cm−1
was recorded by using the IFS 113v Bruker spectrometer. Size
exclusion chromatography (SEC) experiments were performed at 20
°C using a Waters WISP 717 autosampler, a Waters 515 HPLC pump,
and a PSS-SDV 300 × 8 mm column with a particle size of 10 mm and
a pore size of 500, 105, and 106 Å. A RI detector from SOMA and a
UV detector from ERC were used for detection. THF was used as a
solvent. For nuclear magnetic resonance (NMR) analysis H NMR
spectra were recorded on a Bruker Avance 300 spectrometer operating
with a 300 MHz frequency. The polymers were dissolved in an
appropriate solvent. The spectra were calibrated against the solvent
peak. The amount of carboxylic groups was calculated from the results
of the titration experiments performed on a particle charge detector
1
(Mutek GmbH, Germany) in combination with a Titrino automatic
̈
titrator (Metrohm AG, Switzerland). The carboxylic groups were
titrated against the positively charged polycation poly-
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(diallyldimethylammonium chloride) (poly-DADMAC). The titration
was performed using 10 mL of the dispersion with a solid content of 1
g/L. The amount of groups per nm2 was calculated from the
consumed volume of the polyelectrolyte solution. The fluorescence
intensities were measured using a plate reader (Infinite M1000, Tecan,
Switzerland). Mass spectroscopy and ESI mass spectra were obtained
from a Q-ToF Ultima 3 spectrometer with LockSpray interface from
Micromass (Waters). The sample was dissolved in methanol with a
concentration of about 1 mg/mL.
RESULTS AND DISCUSSION
■
Synthesis of PBCA/P(BCA-co-PCA) Nanocapsules.
PBCA nanocapsules containing PBS buffer as aqueous core
were synthesized via anionic polymerization of BCA or a
mixture of BCA and PCA at the water/oil miniemulsion
droplets’ interface. The oil phase consists of Miglyol 812N
(density 0.95 g cm−3, viscosity 30 mPa·s) and a mixture of
surfactants Span80 and Tween80. Different amounts of
monomers BCA, BCA/PCA (in total 0.94 mmol), and
dispersed phase, i.e., PBS buffer solution, were used in order
to study their influence on the physicochemical parameters of
the obtained capsules (Table 1). No precipitation, coagulation,
or flocculation of the nanocapsules was observed throughout
the experiments. After the synthesis and redispersion of the
nanocapsules in an aqueous medium they were analyzed in
terms of average size, size distribution, morphology, and
molecular weight of the formed polymer. The obtained results
are summarized in Table 1. SEM studies (Figure 1A,B) reveal
that the obtained capsules are in a size range between 70 and
150 nm. Moreover, the capsules shell is quite stable and did not
lead to the collapse of the capsules upon drying. Transmission
electron images clearly identify the capsule (core−shell)
morphology (Figure 1C).
The size of the capsules measured by DLS is slightly higher
than that measured by microscopy (Table 1). The hydro-
dynamic size of the capsules was in the range between 235 and
350 nm with a size distribution between 24% and 33%, which is
in the range of an inverse miniemulsion system. When using 1.0
g of PBS buffer, a decrease in the initial BCA/PCA monomer
ratio results in the reduction of the capsule size. The size of
nanocapsules remained approximately the same when using 2.0
g of PBS buffer as dispersed phase. The molecular weight of the
obtained (co)polymer decreases with the decrease in the initial
BCA/PCA monomer ratio. The capsules with the highest
molecular weight were obtained from pure BCA and using 2.0 g
of PBS as dispersed phase.
Figure 2. FTIR spectra of PBCA and P(BCA-co-PCA) nanocapsules
synthesized using 1.0 g (A) or 2.0 g (B) of PBS buffer as dispersed
phase.
bonds as well as to show the simplicity of the described click
procedure.
The Anth-N3 click reaction was performed to study the
formation of the covalent bond between the azide group of the
anthracene and the alkyne groups of the nanocapsules’ surface.
Because of the enhancement of the fluorescent intensity the
formation of a covalent attachment of the dye was proved by a
fluorogenic reaction. The fluorescence of anthracene azide is
quenched due to a photoinduced electron transfer (PET)
process which is not the case in the formation the triazole,
resulting in a 48-fold increase of quantum yield.43 From the
fluorescent enhancement Ω (eq 1), the amount of reacted azide
groups (eq 2) can be calculated. The obtained values are
summarized in Table 2.
The chemical composition of PBCA and P(BCA-co-PCA)
nanocapsules was studied by FT-IR spectroscopy. The obtained
spectra are shown in Figure 2.
The region between 3000 and 1100 cm−1 is known as the
functional group region. In all spectra following bands were
found: 2900 cm−1 (CH2 group), at 2250 cm−1 (−CN group),
1730 cm−1 (CO), and 1100 cm−1 (−C−O group). In the
FT-IR spectra of P(BCA-co-PCA) nanocapsules, an additional
band at 2130 cm−1, which is characteristic for the alkyne bond
from the PCA comonomer, could be seen.
I
I0
Ω =
(1)
⎛
⎜
⎞
⎟
Ω
48
nreact‐Azido
=
n
(Anth‐N3)
⎝
⎠
(2)
Click Reaction of Rho-N3 and Anth-N3 to P(BCA-co-
PCA) Nanocapsules. Although the FT-IR studies confirm that
the P(BCA-co-PCA) capsules contain the alkyne bond, it is still
important to know how many of these groups are able to
participate in the click reaction. Therefore, click reactions of
Rho-N3 and Anth-N3 to P(BCA-co-PCA) nanocapsules were
performed for quantitative determination of the surface alkyne
The introduction of PCA as a comonomer results in the
increase of fluorescence enhancement value. The amount of
covalently attached Anth-N3 onto the capsule surface is slightly
higher when 2 g of the disperse phase was used, and it increases
proportionally to the introduced PCA amount (Figure 3 and
Table 2).
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surface (not covalently bonded) and were not removed by the
centrifugation.
Table 2. Results of Fluorescent Measurements Obtained
After Click Reaction with Anth-N3 and Rho-N3
Synthesis of the Azide-Functionalized Polyurethane
Nanocapsules. An interfacial polyaddition reaction using the
inverse miniemulsion technique was employed to synthesize
the azide-functionalized polyurethane nanocapsules. The
reaction between OH groups from 2,2-bis(azidomethyl)-1,3-
propanediol and NCO groups from TDI occurs at the water-in-
oil droplets’ interface and results in the formation of a cross-
linked polymeric shell. The formulation process is illustrated in
Scheme 6. After redispersion of the capsules in the aqueous
phase, some amounts of azide groups are present on the surface
and could be used for further surface modification through click
chemistry.
amt of alkyne groups per capsule/
a
per nm2
sample
Ω
nreac‑Azido, mmol
Anth-N3
Rho-N3
GB-cc-1
GB-cc-2
GB-cc-3
GB-cc-4
GB-cc-5
GB-cc-6
GB-cc-7
GB-cc-8
1.0
2.4
3.4
4.4
1.1
4.7
5.9
7.9
0.001 17
0.001 66
0.002 15
3.44 × 105/1.4
4.84 × 105/2.8
6.14 × 105/3.7
4.18 × 105/1.7
6.05 × 105/3.5
7.14 × 105/4.3
0.002 30
0.002 88
0.003 86
5.78 × 105/1.5
8.33 × 105/2.3
11.58 × 105/3.2
7.70 × 105/2.0
11.94 × 105/3.3
14.84 × 105/4.1
a
The values are calculated from the fluorescence measurements data,
obtained after click reaction with Rho-N3 or Anth-N3.
The azide-functionalized polyurethane nanocapsules were
characterized in terms of size, size distribution (Table 3), and
Table 3. Characterization Data for the Azide-Functionalized
Polyurethane Nanocapsules before and after Click Reaction
(BAP to TDI Molar Ratio Was 0.9 to 1.0)
zeta
amt of alkyne dye
per capsule/
per nm2
amt of carboxylic
groups per capsule/
per nm2
diam, nm/ potential,
sample
STD, %
mV
GB-MS-
100
285/29
−26
GB-MS-
100-
PA
300/34
310/29
−40
−15
2.22 × 105/0.78
2.02 × 105/0.67
GB-MS-
100-
Alexa
2.15 × 105/0.71
morphology. SEM studies reveal “core−shell” morphology for
the obtained capsules. The collapses of the nanocapsules’ shell
is due to the drying effects and electron beam. The collapse of
the capsules was not observed in the case of PBCA and P(BCA-
co-PCA) capsules (see Figure 1), which might be due to the
formation of a thicker and more stable poly(cyanoacrylate)
shell.
Click Reaction of Propiolic Acid (Cu-Free Reaction)
and of an Alkyne Dye to Azide-Functionalized Polyur-
ethane Nanocapsules. Two different click reactions between
the azide-functionalized polyurethane nanocapsules and either
propiolic acid or alkyne fluorescent dye were performed to
show the versatility in the functionalization (Scheme 7).
In the first reaction, azide-functionalized polyurethane
nanocapsules were functionalized with propiolic acid (sample
GB-MS-100-PA) by Cu-free click chemistry. The reaction was
performed under normal ambient conditions, without addi-
tional heating and without using a catalyst, thus making the
system interesting for various applications, where the presence
of Cu residues affects the properties of the material. The
reaction conditions can be applied for many other alkyne-
functionalized molecules, resulting in the formation of
nanocapsules with variable functionalities.
Figure 3. Fluorescence intensity of Rho-N3 (open symbols) and
fluorescence enhancement of Anth-N3 (solid symbols) after click
reaction.
In further experiments, the fluorescent dye rhodamine azide
was covalently attached to the surface of P(BCA/PCA)
capsules to study the influence of the used amounts of BCA/
PCA on the resulting numbers of alkyne groups on the
nanocapsules’ surface. After the click reaction, as described in
the Experimental Section, the PBCA and P(BCA-co-PCA)
nanocapsules were subjected to the fluorescence measurements.
The results are shown in Figure 3 (samples synthesized using
1.0 and 2.0 g of PBS buffer).
A steeper slope was obtained when 2.0 g of PBS buffer was
used as a disperse phase. Assuming that the density of alkyne
groups remains constant for a given amount of PCA, the
number of surface groups depends on the diameter of the
nanocapsules. Using 1.0 g of PBS buffer, the diameter decreases
by increasing amount of PCA, while remaining constant when
2.0 g of PBS buffer was used. The graphs obtained from the
experiments of the click reaction between Rho-N3 and the
alkyne-functionalized P(BCA-co-PCA) nanocapsules show the
same shape like the graphs obtained from the experiments of
the click reaction between Anth-N3 and the alkyne-function-
alized P(BCA-co-PCA) nanocapsules. From the fluorescent
data, the number of alkyne groups per capsule and nm2 was
calculated (Table 2). The values obtained from the reaction
with Rho-N3 are slightly higher as compared to those with
Anth-N3. This could be explained by the presence of some
Rho-N3 molecules that are strongly adsorbed to the capsule’s
In the second click reaction, fluorescent alkyne dye was
reacted with azide-functionalized polyurethane nanocapsules
(sample GB-MS-100-Alexa) in order to demonstrate the
possibility of fluorescent labeling of nanocapsules that could
be used for trafficking. Before addition of CuAAC, the
fluorescent signal (λex = 488 nm, λem = 520 nm) was measured
and set as a reference signal. After click reaction the
nanocapsules were centrifuged and the fluorescent signal of
the supernatant was measured and compared to the reference.
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Scheme 7. Surface Click Reaction between Azide-Functionalized Polyurethane Nanocapsules and (A) Propiolic Acid (Cu-Free
Reaction) or (B) Alkyne Fluorescent Dye
The obtained results reveal that about 94% of the alkyne dye
was attached to the nanocapsules surface. The calculated
amount of alkyne dye molecules per capsule and per nm2 are
given in Table 3.
The average diameter of capsules before click reaction was
about 285 nm. A slight increase of size and size distribution was
observed after the click reaction, confirming the presence of
“clicked” molecules on the surface. Both clicking procedures
did not influence the capsule’s morphology as it could be seen
from the SEM images of nanocapsules made after the reactions
(Scheme 7). The zeta potential values after click reaction are
different from the nonfunctionalized capsules. The decrease
(sample GB-MS-100-PA) or increase (sample GB-MS-100-
Alexa) of value mainly depends on the charge of molecules that
were used for the click reaction. The amount of carboxylic
groups that are present on the nanocapsules surface was
calculated from the particle charge detector (PCD) measure-
1
Figure 4. H NMR spectra in d6-DMSO of the Cu-free click reaction
between 2,2-bis(azidomethyl)-1,3-propanediol and propiolic acid after
certain time periods.
ments. The determined amount of carboxylic groups was
slightly higher when the surface of capsules was functionalized
with the propiolic acid.
Investigation of the Cu-Free Reaction Kinetics
between Propiolic Acid and Azide Monomer. The
reaction kinetics between the propiolic acid and the azide
monomer (2,2-bis(azidomethyl)-1,3-propanediol) was studied
by NMR spectroscopy in DMSO. The results are presented in
Figure 4.
It can be seen that the characteristic peak from the used
monomers (3.28 ppm) decreases, and the new peaks
corresponding to triazole ring evolve. Furthermore, two new
signals between 8.45 and 8.55 ppm were observed. These are
originated from the proton of the C−C double bound of the
triazole ring. The reaction proceeded smoothly under ambient
conditions within 48 h. A 1,4-isomer as well as an 1,5-isomer
was found.
CONCLUSION
■
In this work, we have demonstrate for the first time the ability
to prepare polycyanoacrylate- and polyurethane-based nano-
capsules with several types of surface functionalization, while
utilizing different Cu(I)-catalyzed azide alkyne cycloaddition
(CuAAC) reactions as well as in the absence of copper. In the
case of polycyanoacrylate-based capsules, the density of
functionalization can be adjusted by using different amounts
of propargyl cyanoacrylate comonomer. The results of click
reaction between nanocapsules and anthracene azide reveal that
the amount of surface available propargyl groups increases by a
factor of 0.65 as compared to the introduced amount of
propargyl cyanoacrylate. Click reaction performed on the
surface of polyurethane azide nanocapsules with copper
catalysis (alkyne dye) or under copper-free conditions
(propiolic acid) results in similar functionalization density
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(0.70 vs 0.78 groups per nm2), thus making the described
copper-free reaction promising for the biological applications.
The proposed concept for surface functionalization of
polycyanoacrylate- and polyurethane-based nanocapsules
through click reactions could be extended to many others
alkyne or azide molecules, thus leading to the formation of
novel materials for diverse applications.
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AUTHOR INFORMATION
Corresponding Author
■
*E-mail musyanovych@mpip-mainz.mpg.de, Tel +49(0)6131
379-248, Fax +49(0)6131 379-100.
́ ́ ̌ ́
(29) Mackova, H.; Proks, V.; Horak, D.; Kucka, J.; Trchova, M. J.
Polym. Sci., Part A: Polym. Chem. 2011, 49, 4820−4829.
Notes
(30) Krovi, S. A.; Smith, D.; Nguyen, S. T. Chem. Commun. 2010, 46,
5277−5279.
The authors declare no competing financial interest.
(31) Kamphuis, M. M. J.; Johnston, A. P. R.; Such, G. K.; Dam, H.
H.; Evans, R. A.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. J.
Am. Chem. Soc. 2010, 132, 15881−15883.
ACKNOWLEDGMENTS
We thank Elke Muth, Gunnar Glasser, and Christoph Sieber for
their help in FTIR, TEM, and SEM measurements. We also
■
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