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Morphology control and surface functionalization of

proteinSiO2 hybrid capsules
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Cite this: J. Mater. Chem. B, 2013, 1,

6427
Huihui Wang,a Tayebeh Mirzaei Garakani,a Tim Krappitz,a Patrick van Rijn*ab
ker*a
and Alexander Bo

Received 22nd July 2013
Accepted 4th October 2013
DOI: 10.1039/c3tb21013h
www.rsc.org/MaterialsB
In this contribution, we describe ways to introduce additional complexity and functionality to protein
mediated capsule formation based on biomineralization in Pickering templated systems in order to
enable possible post-mineralization modications. Here the shell morphology is inuenced by addition
of ionic additives to the reaction system which signicantly alters the surface structure. By changing the
oil-phase (tetraethyl orthosilicate), even more complexity is introduced as well as reactive groups by
adding (3-aminopropyl)trimethoxysilane to the oil phase. The incorporated amino-functionality is easily
addressed via mild peptide coupling reaction.

Introduction
The superior properties of natural materials are oen based on
the hybrid character of these structures in which proteins fulll
a key role.1 Particularly interesting is the fact that highly useful
properties can be introduced via proteins which arise from their
specic size, catalytic activity, coordinating ability and behavior
similar to colloidal particles.2,3 Especially, in the process of
biomineralization,4,5 the formation of the mineral phase and
the mechanical properties arising from the dened internal
structure6 are due to a combination of those properties.79 These
fascinating materials inspire to use similar approaches which
led to the formation of various synthetic inorganicprotein
hybrid structures.1012 Hydroxyapatite (CaPO4),13,14 calcite
(CaCO3)15 and silicon dioxide16 are the most frequently used
minerals in these hybrid structures but also other oxides like
TiO2, ZrO2 and GaOOH were employed.17,18 Hydroxyapatite and
CaCO3 have the advantage of being native components of the
body while SiO2 is used because of the convenient semi-organic
precursors available which are converted by various hydrolases,
e.g. silicatein,19 trypsin20 and lysozyme.21 The formation of
silicon dioxide structures via biocatalysis has drawn much
a
DWI an der RWTH Aachen e.V., Lehrstuhl fur Makromolekulare Materialien und
Ober
achen, RWTH Aachen University, Forckenbeckstrasse 50, D-52056 Aachen,
Germany. E-mail: boker@dwi.rwth-aachen.de; Fax: +49-241-8023317; Tel: +49-241-
8023304
b
Department of Biomedical Engineering-FB40, W.J. Kol Institute for Biomedical
Engineering and Materials Science, University of Groningen, University Medical
Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail:
p.van.rijn@umcg.nl; Tel: +31-50-3633141
Electronic supplementary information (ESI) available: Interfacial tension
measurements, absorption characterization and optical/uorescence
micrographs. See DOI: 10.1039/c3tb21013h
These authors contributed equally.
attention not only due to the use of enzymes22,23 but also with
respect to small peptide fragments e.g. for silicon dioxide
surface coatings.24 Protein-mediated SiO2 formation under near
physiological conditions has high potential in the eld of tissue
engineering, for example as novel implant materials,25 or in the
immobilization of sensitive catalysts26 or enzymes.27 Methods
have been reported to prepare monodisperse silica capsules via
a two-step solgel process with tetraethyl orthosilicate (TEOS)
using various templating methodologies e.g. by emulsion or
polymeric aggregates.6,28,29 However, TEOS can also be con-
verted via a polycondensation reaction into SiO2 by lysozyme
which is attractive because lysozyme is commercially available,
inexpensive and displays higher interfacial activity towards
polarapolar interfaces than other commercially available
hydrolases which is an advantage in Pickering emulsion tem-
plated systems (Fig. S1) leading to the formation of hollow
SiO2lysozyme hybrid capsules.30 This approach has recently
been ne-tuned in order to gain more control over the hybrid
morphology and in addition to nanoparticles and nano-/
microcapsules also inverted capsules and double emulsion
capsules have been prepared.31 While the formation of various
capsules is important for the type of application, additional
control over the shell morphology as well as the incorporation
of functionality and reactivity would make the system more
versatile.
Here we present an easy approach to inuence the shell
morphology and reactivity without changing the preparation
method. This is achieved by changing the buer type and
composition used as the aqueous phase or by changing the
silicon dioxide precursor composition. Changing the buer
composition with the addition of various salts or ionic surfac-
tants changed the capsule shell structure. Changing the SiO2-
precursor also inuences the shell and when (3-aminopropyl)
trimethoxysilane (APTMS) is used in minor amounts, the shell

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Journal of Materials Chemistry B Paper

surface possesses primary amine-functionalities which were
modied via mild peptide-coupling reactions for which we used
uorescent molecular dyes as model compounds.
Experimental part
Materials
All reactants (TEOS, APTMS, lysozyme, glycine/PBS (pH 9.0))
were obtained from Sigma Aldrich and used without any further
purication. PEOS was kindly provided by Dr Xiaoming Zhu,
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aqueous capsule suspension. The reaction mixture was shaken
at 37  C for 24 h and puried by centrifugation, decantation and
addition of water.
Dabsyl addition. 100 mL dabsyl chloride solution in dime-
thylformamide (DMF) (5 mg mL 1) was added to 900 mL of
sample solution. The reaction mixture was shaken at 37  C for
24 h and the product was washed with DMF and water by
centrifugation, decantation and addition of water.
Results and discussion

DWI an der RWTH and prepared according to previously

reported synthetic procedures.32 The changes in the interfacial
For the SiO2lysozyme structures either the conventional
tension between the protein solution and the oil phase were
approach (CA) or the recently described two-step TEOS addition
determined at room temperature and monitored with a DSA100
procedure (TSA) was used. In a previous study it was identied
pendant-drop tensiometer with a CCD-camera for drop-image
that lysozyme mostly retains its 3-dimensional structure
processing, which allows rapid drop-image acquisition, edge
according to Raman spectroscopy.31 Changing the convention-
detection and tting of the YoungLaplace equation. Small
ally used glycine buer to phosphate buer saline (PBS),
blunt-end cannulas of 1.83 mm in diameter (NE45 Kr uss) were
changes were introduced in the shell surface structure for both
used with 100 mL min 1 for the speed of drop-formation. A
the CA and the TSA method. The use of glycine buer resulted
Bandelin Sonorex operating at 35 kHz at ambient temperature
in capsules with a broad size distribution (300 nm to 15 mm, CA)
was used for the sonication treatment. SEM analysis was per-
(Fig. 1A) and small capsules (<2 mm, TSA) both with a rather
formed on HITACHI S-3000N and HITACHI S-4800 scanning
smooth shell (Fig. 1C), while with PBS considerably bigger
electron microscopes and the EDX data were taken using an
capsules were formed (530 mm, CA; 15 mm, TSA) (Fig. 1B
EDAX detecting unit. TEM analysis was performed on a ZEISS
and D) with a rough shell on which small particles are seen.
LIBRA120 PLUS electron microscope, operating at 120 kV.
From the EDX analysis of puried capsules (Fig. 2) signals of Na
Fluorescence and optical microscopy were performed with a
and P are detected indicating that these are incorporated from
Keyence Biozero (BZ-8100E) microscope, with excitation-mode
the buer into the lysozymeSiO2 hybrid capsules while these
Texas Red as well as GFP for visualization of calcein. The UV-Vis
elements were not detected in the glycine buer capsules.
measurements were done with an EVOLUTION 300 UV-Visible
The buer-type aects the morphology of the hybrid struc-
spectrometer.
ture, which is anticipated to originate from the dierence in
ionic strength of the buer since this is the major dierence
General capsule preparation
between glycine buer and PBS. According to EDX, Na and P
Conventional Approach (CA). 900 mL of lysozyme solution were incorporated while K is not detected which is the second
(5 mg mL 1, 0.34 mM) in the respective buer (glycine or PBS) most abundant cation present in PBS. Therefore, it was expec-
and 100 mL TEOS (or PEOS) were mixed via sonication (15 min, ted that other additives in the form of sodium salts also aect
35 kHz) and allowed to react at room temperature for 24 h, the capsule formation considerably. The same synthesis was
providing a mineralized emulsion droplet. To achieve the performed in glycine buer (TSA) but with the additives: NaCl
reactive capsules, which have amino-functionalities incorpo-
rated into the shell, a mixture of TEOSAPTMS (92/8 volume
ratio; 8.9/1 molar ratio) was used instead of only TEOS.
Two-Step Addition (TSA). 20 mL TEOS (or PEOS) was added to
900 mL of lysozyme solution (5 mg mL 1) in the respective buer
(glycine or PBS) and then stirred gently (5 rpm) for 30 minutes.
Aer the rst addition, the remaining 80 mL TEOS was added
and sonication (35 kHz) was applied for 15 minutes followed by
gently stirring for 24 hours at room temperature (5 rpm) and
nalized again with sonication (15 min, 35 kHz). For the
experiments where ionic additives NaCl (2.9 mg, 0.5 mmol), NaF
(2.1 mg, 0.5 mmol), CTAB (2.1 mg, 6 mmol) or SDS (1.7 mg,
6 mmol) were incorporated, the additives were introduced
during the second addition of the silicon dioxide precursor.

Synthetic modication of capsules

Fig. 1 SEM images of SiO2lysozyme capsules prepared via the CA method using
Peptide coupling. For the reaction with the uorescent dye glycine buer (A) and phosphate buer saline (B) and prepared via the TSA
calcein, 1 mg calcein together with 1 mg N-(3-dimethylamino- method also in glycine buer (C) and phosphate buer saline (D). Insets show
propyl)N-ethylcarbodiimide hydrochloride was added to 1 mL magnications of the surface of the capsules.

6428 | J. Mater. Chem. B, 2013, 1, 64276433 This journal is The Royal Society of Chemistry 2013

Paper
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Fig. 2 EDX measurements, giving the binding energies in eV, performed on the
corresponding SEM samples shown in Fig. 1 display the presence of no additional
ions for the glycine buer (A) and Na and the corresponding counter-ions for
Na2HPO4 (B), NaCl (C) and NaF (D).
and NaF. By introducing NaCl (2.9 mg, 0.5 mmol) the size of the
capsules increases signicantly to about 510 mm (Fig. 3A).
Obviously, NaCl has inuence on the TEOS/lysozyme emulsion,
especially on the drop size of the dispersed phase. As the
positive charges in lysozyme cause electrostatic repulsion
between the droplets, it makes the emulsion more stable, which
is most likely reduced by ionprotein interactions screening the
charges.33 The chloride ions bind to lysozyme and reduce the
electrostatic repulsion between the TEOS droplets, which
facilitates coalescence, giving bigger capsules. Adding NaF
Fig. 3 SEM images of SiO2lysozyme capsules prepared via the TSA method
using glycine buer and various additives like NaCl (A), NaF (B) and CTAB (C). In (D)
the corresponding TEM analysis of (C) is shown.
This journal is The Royal Society of Chemistry 2013
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Journal of Materials Chemistry B
(2.1 mg, 0.5 mmol) decreases the amount of capsules signi-
cantly and provides even larger capsules (>20 mm) with a fragile
shell (Fig. 3D). NaF seems to have much stronger inuence than
NaCl on the stability of the TEOS/lysozyme emulsion probably
due to a higher charge density of the uorine ions compared to
the chlorine. The catalytic activity of lysozyme seems not to be
aected since similar amounts of SiO2 were isolated for all
reactions. In all cases the sodium salt with the corresponding
counter ion is incorporated into the SiO2lysozyme hybrid
structure as shown by EDX (Fig. 2). However, in the case of NaF,
the incorporation is only marginal and although a low EDX-
signal for Na was found, F could not be detected. This could also
be due to the dierence in relative association strengths of F
and Cl towards Na or the cationic amino acids of the lysozyme
(mostly lysines). The relative anity of F for Na compared to
positively charged lysine is higher than that of Cl towards Na
and lysine. Hence more NaCl is incorporated and in the NaF
addition only Na is detected since the anity of Na towards
negatively charged silicate of the capsule surface is higher than
towards F and therefore Na is conned to the surface of the
capsules and F is not detected in the EDX. This suggests that
several interactions play a role and that the overall system is
more complex than simple anionprotein interactions.
It is known that specic ion-coordination to polypeptides
and polyamines is a major factor aecting the rate of SiO2
formation. More importantly it also inuences the morphology
of the SiO2 structure.34 As lysozyme is positively charged35 and
SiO2 species are negatively charged,36 the electrostatic interac-
tions and/or other interactions of the anions with lysozyme and
SiO2 also aect the sizes and morphologies of the proteinSiO2
hybrid capsules. However, the salts destabilize the emulsion
and hence an ion-pair was chosen with additional stabilizing
features to circumvent the loss of emulsion stability. An anionic
surfactant sodium dodecylsulphate (SDS) as well as a cationic
surfactant cetyltrimethylammonium bromide (CTAB) was used
as an additive both at 6.67 mM. SDS and CTAB were used to
monitor the inuence of charge and SDS is promising since this
is also a sodium salt. Although the critical micelle concentra-
tion (cmc) diers signicantly between the two, 1 mM for CTAB
and 710 mM for SDS, this can be neglected since surfactant
aggregates and monomers are in a dynamic equilibrium. This
means that aer adsorption of a monomer surfactant in solu-
tion onto the interface, the loss of monomer is compensated by
the dis-assembly of micellar aggregates. So even though the
concentration of SDS is below the cmc, it should not aect
the emulsication properties as compared to CTAB of which the
concentration is above the cmc. The addition of SDS did not
result in capsule formation and only unidentied liquid-like
structures were obtained. This is probably caused by the coor-
dination of SDS to lysozyme via ionic interactions similar to that
for the other anions. It is known that SDS associates to lysozyme
up to a molar ratio of 100 : 1 (SDS : lysozyme) before signi-
cantly denaturing the protein and coordination occurs between
the positively charged amino acids on the surface and the
negative charge of the SDS.37 This forms an apolar layer around
the lysozyme. Although this still enables the complex to stabi-
lize the interface, SDS is much bigger than the other anions and
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the hydrophobic tails of SDS surrounding the lysozyme can

shield the enzyme, inhibiting the catalytic activity and hence
prevent SiO2 formation. Both SDS as well as the lysozymeSDS
complex is able to stabilize the interfaces and hence emulsi-
cation properties are maintained. Upon addition of CTAB
(2.1 mg, 6 mmol), nanocapsules are observed with diameters
between 200 and 600 nm (Fig. 3C and D). The silicication
process is not compromised by the addition of the surfactant
and the additional stabilizing eect of the CTAB provides
smaller capsules with a smooth and rather thick shell. Addi-
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tionally, a large amount of SiO2 nanoparticles of about 50 nm in

diameter were formed which were observed along with the
capsules by TEM analysis (Fig. 3D). CTAB by itself forms
micellar aggregates and can act as a template for SiO2 deposi-
tion and because the concentration of CTAB used in the
experiment is about 7 times higher than the cmc, a large
amount of micelles and hence smaller nanostructures can be
expected.38
In emulsion templating, TEOS is condensed and polymerizes
to SiO2 and we envisioned that changing the silicon dioxide
precursor is also a way of aecting the morphology as well as
incorporating reactivity. Using a liquid hyperbranched poly-
Fig. 4 SEM images of SiO2lysozyme capsules prepared via the TSA method
ethoxysilane (PEOS), it was anticipated that the reaction would using glycine buer (AC) and phosphate buer saline (DF) with additives NaCl
be more ecient since less condensation needs to take place. (A and D), NaF (B and E) and CTAB (C and F).
However, in the end no capsules were found via the CA and TSA
method in glycine buer as well as in PBS, which was rather
unexpected. Probably PEOS is too large and is sterically performed using the same conditions without lysozyme
hindered to reach the reactive center of the lysozyme. However, (Fig. S2). Capsules with only CTAB were formed but were
this seems unlikely since PEOS represents an advanced state in smooth and very thin displaying collapsed capsule structures.
the condensation reaction which is ultimately reached also with SiO2 surfaces can be easily modied using the St ober
TEOS and silicon oxide is being formed using PEOS only not in a method.39,40 In the next section, we show that the incorporation
capsule form but as a bulk precipitate. The other aspect of PEOS of an additional functionality can also be performed using
is that it is much more viscous than TEOS and it was thought enzymes at room temperature, so that the typical St ober
that by using a mixed precursor composition by rst adding conditions, i.e. ammoniaethanol mixtures at 80  C, can be
PEOS to the system and secondly TEOS it would facilitate the replaced by a mild procedure. Therefore, we added small
mineralization process and that by using the previously amounts of APTMS to the TEOS in the CA method in glycine
mentioned additives, the shell structure could be inuenced buer, providing an amine-functionality on the capsule surface
even more. in one step, which was used directly for further chemical
The TSA method with rst adding 20 mL PEOS and secondly modication. Various APTMSTEOS ratios were used and it was
80 mL TEOS without any additives did not result in capsule found that with TEOS contents below 90%, a gelled reaction
formation. However, with additives not only capsules were mixture was obtained. Only above 90% TEOS, dispersible
formed but also altered surface morphologies were displayed structures were obtained and 92/8 was the highest ratio which
compared to the use of TEOS only. Adding NaCl to the system in still provided capsule structures (Fig. 5A and B) which were
glycine buer resulted in perfectly smooth and nicely separated
capsules. In PBS the capsules are fused together (Fig. 4A and D).
Adding NaF to the system in glycine buer resulted in similar
capsules to those for NaCl (Fig. 4B) but in PBS the capsules are
well separated and have a very smooth shell structure (Fig. 4E).
The most striking change in surface morphology was observed
for the addition of CTAB. While before with TEOS only, nano-
capsules with a very smooth shell structure were obtained, and
when used in combination with PEOS/TEOS, a very folded and
crumpled structure is formed (Fig. 4C and F). In glycine buer
the features are very pronounced (Fig. 4C) while in PBS the
wrinkles are attened but still visible (Fig. 4F). Using SDS still
did not result in capsule formation. To show that the structure Fig. 5 SEM (A) and TEM (B) image of SiO2NH2lysozyme capsules prepared via
is not induced solely by CTAB, a control experiment was mixing of TEOS and APMTS (92/8) using the CA method.

6430 | J. Mater. Chem. B, 2013, 1, 64276433 This journal is The Royal Society of Chemistry 2013
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Paper Journal of Materials Chemistry B

puried by washing the solid phase several times via decanta-

tion. The samples were additionally washed with ethanol and
water to remove possible remaining APTMS and the capsules
were tested for reactivity towards the amine functionality.
Dabsyl chloride and calcein were chosen to determine the
presence of the primary amine groups. Dabsyl chloride reacts
readily with primary amines while for calcein a carbodiimide
coupling reagent (EDC) is needed, which represents a standard
peptide coupling reaction (Fig. 6 and 7). 100 mL dabsyl chloride
solution in dimethylformamide (DMF) (5 mg mL 1) was added
to 900 mL of sample solution. The reaction mixture was shaken
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at 37  C for 24 h and the product was washed with DMF and

water. In order to identify the exact nature of the reaction,
several control reactions were performed under similar condi-
tions with silicon dioxide nanoparticles, APTMS functionalized
silicon dioxide nanoparticles, pure lysozyme and SiO2lysozyme
capsules. Each condition provides insight into which structures
are reactive and as to whether the origin of the reactivity is due
to the added functionality or also possibly via a reaction with
SiO2, lysozyme or due to physisorption onto the structures. All
products were analyzed by absorption spectroscopy aer dial-
ysis against DMFwater (Fig. S3). In the case of pure silica
nanoparticles, no dabsyl absorption was detected while APTMS
modied nanoparticles as well as pure lysozyme do show a
specic absorption, indicating a reaction with dabsyl chloride.
The conventional SiO2lysozyme capsules show no absorption
while the capsules modied with APTMS show a shoulder at 465
nm indicative for dabsyl. This indicates successful functional-
ization of the capsules with APTMS and that there is no reac-
tivity with respect to functionalization towards the incorporated
proteins, i.e. we may conclude that the enzyme does not reside
at the capsule surface.
For the reaction with the uorescent dye calcein, 1 mg cal-
cein together with 1 mg N-(3-dimethylaminopropyl)N-ethyl-
carbodiimide hydrochloride was added to 1 mL aqueous
capsule suspension. Again the reaction mixture was shaken at
37  C for 24 h and puried by centrifugation. We compare the
SiO2NH2lysozyme capsules in which APTMS was added to the
Fig. 6 UV-Vis spectra of pure calcein as well as calcein-modied conventional
SiO2lysozyme capsules and SiO2NH2lysozyme capsules via EDC-induced
peptide coupling (measured in H2O).
Fig. 7 Fluorescence microscopy images in the bright eld (A and D), green (B and E)
and red (C and F) channels of the conventional SiO2lysozyme capsules (AC) and
SiO2NH2lysozyme capsules (DF), both were treated with calcein/EDC.
oil-phase with conventional SiO2lysozyme capsules and SiO2
NH2 nanoparticles aer the same treatment with calcein. Aer
the reaction and purication by several washing and centrifu-
gation steps, all three samples remained colored indicating that
calcein binds to all. However, the supernatant of the APTMS
modied nanoparticles did not display any color aer centri-
fugation while for both capsule systems there is continuous
coloring of the supernatant aer each washing indicating that
there is physisorption of the dye, probably due to the positive
charges of the lysozyme interacting with the negative charges of
the calcein (Fig. S4). For the APTMS capsules the supernatant
coloring became less and resulted in colored capsules indi-
cating covalent binding, while the conventional capsules
continued to lose color. The covalent binding was also indicated
by absorption measurements. A bathochromic shi in absorp-
tion was observed probably due to inter-molecular p-interac-
tions/stacking in the covalently bound system as compared to
the physisorbed one, indicating a high surface concentration of
calcein (Fig. 6). Such bathochromic shis were also observed in
other calcein-based systems.41
The bathochromic shi also explains the dierence in uo-
rescent behavior, which was observed in the uorescence
microscopy analysis of the conventional silicalysozyme
capsules and the amine-functionalized capsules (Fig. 7). For the
capsules onto which the calcein is physisorbed, the uores-
cence is detected in the green which is normally the case for
calcein while in the red no signal is observed. In the case of the
covalently bound system, the aqueous phase is green, presum-
ably originating from still desorbed calcein moieties while the

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