Formation of cage-like hollow spherical silica via a mesoporous structure by calcination of lysozyme-silica hybrid particles

Article abstract of DOI:10.1039/b709534a

Calcination of lysozyme-silica hybrid hollow particles gives novel cage-like hollow spherical silicas with differently patterned through-holes on their shell structure. The Royal Society of Chemistry.

Full text of DOI:10.1039/b709534a

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Formation of cage-like hollow spherical silica via a mesoporous
structure by calcination of lysozyme–silica hybrid particles{
Toru Shiomi,ab Tatsuo Tsunoda,*b Akiko Kawai,b Fujio Mizukamib and Kengo Sakaguchi*a
Received (in Cambridge, UK) 22nd June 2007, Accepted 10th August 2007
First published as an Advance Article on the web 21st August 2007
DOI: 10.1039/b709534a
morphology of the silica structure. The removal of organic
templates by calcination from organic–inorganic hybrid materials
is a general technique to transfer the diverse structure of organic
molecular to inorganic products.7 Several examples of templated
formation of inorganic materials have employed biopolymers such
as DNA, peptide fibrils and polysaccharides, whose characteristic
shapes have been faithfully transferred to inorganic materials.8
However, our synthesis route to CHS is somewhat different from
the transcription method using a bioorganic polymer just as a
template, because the through-hole sizes of the CHS synthesized in
this study were ten times larger than the molecular size of
lysozyme. This difference is an interesting hint towards under-
standing the formation process of CHS. Based on the observation
of the morphologies at each calcination temperature, we will also
discuss the formation mechanism of through-holes whose sizes are
larger than that of a lysozyme molecule.
Calcination of lysozyme–silica hybrid hollow particles gives
novel cage-like hollow spherical silicas with differently
patterned through-holes on their shell structure.
The controlled formation of inorganic materials with well designed
shapes and patterns at the micron- and nano-size level is extremely
important in materials science. In particular, the preparation of
hollow inorganic capsules with a defined structure has received
increasing attention, because of their potential applications, such
as the encapsulation of various substances, as controlled release
systems for drug delivery and the manufacture of advanced
materials.1 The key to using hollow inorganic shells in these
applications is the design of the interior space as a storage space or
a reaction chamber and the shell structure containing paths
through which desired molecules can be loaded or released.2 Many
researchers have found sophisticated methods for creating
inorganic hollow structures with sponge-like interiors, mesoporous
shell structures, micro-spheres with surface perforations or self-
organized porous spherical particles.3 However, to the best of our
knowledge, a hollow spherical structure, with a defined interior
space and a shell provided with through-holes of .100 nm
diameter, sufficiently large to allow the passage of biomacromo-
lecules, is essentially unknown.4 Very recently, silica hollow spheres
with nano-macroholes like diatomaceous earth have been
synthesized using a water/oil/water emulsion system, although
the formation mechanism of the macroholes is still unclear.5 The
generation of capsule shapes with complex microscopic structures
like cell walls remains the ultimate challenge to workers in the field.
Herein we report the morphologies of novel cage-like hollow
spherical silica (CHS) with a through-hole (50–250 nm diameter)
structure obtained by the calcination of lysozyme–silica hybrid
hollow particles (L-SHHs). L-SHHs were synthesized by the
combination of sonochemical treatment and the silica precipitation
activity of lysozyme. Lysozyme molecules were found to be
uniformly dispersed within the silica matrix.6 One can envision the
removal of such encapsulated lysozyme as a way to control the
Our synthesis of CHS consists of two simple steps.{ First,
L-SHHs were prepared by reacting TEOS with 2 mg ml21
lysozyme solution via a sonochemical treatment, as previously
reported.6 The obtained L-SHHs with a particle diameter of 0.5–
15 mm were subsequently calcined at 700 uC for 2 h in air, resulting
in the formation of CHS. Fig. 1 shows scanning electron
microscope (SEM) and transmission electron microscope (TEM)
images of the CHS. The hollow spherical structure of L-SHHs was
preserved even after calcination, and the characteristic through-
holes which are similar to those in an archean radiolarian shell9
were formed in the silica shell structures. The typical CHS
structure has a silica shell with thickness of y100 nm and through-
holes with a diameter of 50–250 nm. The particle size distribution
of typical CHS appeared not to differ from that of L-SHHs (0.5–
15 mm), even though slight shrinkage might occur during
calcination. The through-holes of CHS shell structures were
individually patterned at random even in the same sample. The
morphological variety of the through-hole structure was observed
in relation to the number, size, and shape of through-holes and the
connectivity of adjacent through-holes. It appears that the
through-holes pattern is very sensitive to the structural nature of
the L-SHHs. CHS is mechanically fragile so that fragmentation or
collapse of particles as seen in Fig. 1C easily occurred.
Additionally, the broad range distribution of shell thickness in
L-SHHs should be mentioned. In the case where the shell thickness
is above 100 nm, the alternative shape of CHS shown in Fig. 2 was
also observed. Instead of a thin silica shell layer and complete
through-holes, some CHS have a bilayer shell structure with
complicated voids between silica shell layers (Fig. 2A and C), and
others have a slightly thicker silica shell with incomplete through-
holes (Fig. 2B and D). It appears that one of the essential factors
aDepartment of Applied Biological Science, Faculty of Science and
Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi,
Chiba-ken, 278-8510, Japan. E-mail: kengo@rs.noda.tus.ac.jp;
Fax: +81-471-23-9767; Tel: +81-471-24-1501(ext.3409)
bResearch Center for Compact Chemical Process, National Institute of
Advanced Industrial Science and Technology (AIST), Central5, 1-1-1
Higashi, Tsukuba, Ibaraki-ken, 305-8565, Japan.
E-mail: t.tsunoda@aist.go.jp; Fax: +81-29-861-4633;
Tel: +81-29-861-4633
{ Electronic supplementary information (ESI) available: SEM and TEM
images; nitrogen adsorption and desorption isotherms; BET surface areas
of as-synthesized L-SHHs and L-SHHs calcined at different temperatures.
See DOI: 10.1039/b709534a
4404 | Chem. Commun., 2007, 4404–4406
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Fig. 1 SEM (A), (B) and TEM (C) images of CHS obtained by calcining L-SHHs (2 mg ml21 lysozyme) at 700 uC for 2 h. Scale bars: (A) 3 mm, (B) and
(C) 500 nm.
Fig. 3 SEM (A) and TEM (Inset) and (B) images of CHS obtained by
calcining L-SHHs (10 mg ml21 lysozyme) at 700 uC for 2 h. Scale bars: (A)
and (Inset) 1 mm, (B) 300 nm.
Fig. 2 SEM and TEM images of CHS with different shell thickness. The
CHS structures were also observed in the same sample prepared by
calcining L-SHHs (2 mg ml21 lysozyme) at 700 uC for 2 h. All scale bars:
500 nm.
operating to form a complete CHS shell structure is the shell
thickness of the L-SHHs.
The composition of the L-SHHs is also likely to be another key
factor that controls the variable nature of the patterns of CHS shell
structures. To investigate this, we examined how the initial
lysozyme concentration used to prepare L-SHHs influences the
morphology of the CHS formed after calcination. When the
L-SHHs were prepared with a 1 mg ml21 lysozyme solution,
calcination resulted in a CHS with a similar shape to those made
from 2 mg ml21, while those prepared from a 10 mg ml21 solution
exhibit a different morphology. As Fig. 3A reveals, the SEM and
TEM images of calcined L-SHHs made from a 10 mg ml21
Fig. 4 TGA analysis of L-SHHs obtained with different lysozyme
solution show a significantly rough shell surface with incomplete
concentrations.
through-holes and serpentine wormlike structures. Under the high
respectively. The relatively high lysozyme content of L-SHHs
which were synthesized with 10 mg ml21 lysozyme solution
probably represents the increase of lysozyme ratio within the near
surface area of L-SHHs. This difference would cause the formation
of a rough surface and serpentine wormlike structures.
magnification of the TEM, it was apparent that mesopores with
diameters of several tens of nanometers were formed even on the
inside of the wormlike structures (indicated by an arrow in
Fig. 3B). Thermal gravimetric analysis (TGA) clearly showed that
the different surface morphologies of the calcined L-SHHs are
linked to the composition ratios of lysozyme to silica (Fig. 4). Total
weight losses between 30 and 700 uC of materials made from
2 mg ml21 and 10 mg ml21 solutions were ca. 55 and 63%,
Having established the role of the properties of the L-SHHs in
the calcination process, we went on to investigate the formation
mechanism of CHSs. One aspect of this important topic is the
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with through-holes (Fig. 2A). In order to explain the formation
mechanism of CHS in more detail, further work needs to be
performed on the synthesis method using more precisely-
controlled, well-defined CHSs. Nevertheless, it can be concluded
that the formation process of CHS during calcination is comprised
of two steps: the removal of lysozyme resulting in the formation of
mesopores by the templating effect of lysozyme (first step) and the
subsequent restructuring of the silica network leading to a
morphological change to the CHS (second step).
We have demonstrated that a novel cage-like hollow spherical
silica (CHS) was synthesized by the calcination of lysozyme–silica
hybrid hollow particles (L-SHHs). The shell thickness and the
composition ratio of L-SHHs were key factors in the resulting
CHS structure. Based on the formation of cage-like hollow
spherical particles with a through-hole structure, we suggest not
only that lysozyme molecules operate as a sacrificial organic
template during the calcination of L-SHHs, but also that
reconstruction of the silica structure occurs after removal of the
lysozyme molecules. The through-holes and interior space offered
by the CHS structures have the potential to be developed for
applications such as controlled release systems. The results of our
findings reported herein suggest the possibility of developing a
practical synthesis of cage-like particles and other shaped
nanomaterials using biopolymer–inorganic hybrid structures as
the primary material.
Fig. 5 Pore size distributions calculated from nitrogen adsorption
measurements of as-synthesized L-SHHs and L-SHHs calcined at different
temperatures.
curious question of how the large through-holes diameter (50–
250 nm) of the CHS is created from the small lysozyme molecule
(3.0 nm 6 3.0 nm 6 4.5 nm) encapsulated within the silica matrix
of L-SHHs during calcination. To resolve this question, we
performed nitrogen adsorption–desorption experiments with
samples obtained from the calcination of L-SHHs (prepared with
2 mg ml21 lysozyme solution) at different temperatures.
Fig. 5 shows the pore size distribution calculated by the BJH
method using the adsorption branch. As-synthesized L-SHHs were
found to have no mesopore structure. When calcination was
performed at 400 uC, pores with diameter of about 2 nm or less
were observed. It is worth noting that pores with a diameter of
about 3 nm, which is consistent with the size of the lysozyme
molecule, appeared after calcination at 500 uC. Increasing the
calcination temperature from 500 to 600 uC decreases the size of
the pore distribution peak. The BET surface areas also decreased
with an increase in the calcination temperature from 400 to 600 uC
(621, 420 and 338 m2 g21, respectively). These mesopores
disappeared after calcination at 700 uC (the temperature used in
the preparation of the CHSs), while larger pores appeared in the
range of several tens of nanometers or more, which probably
corresponds to creation of the through-holes observed in CHS.
Returning to the TGA results, Fig. 4 shows that the removal of
lysozyme from L-SHHs finishes at about 500 uC. Thus, the
mesopores of around 3 nm diameter are probably derived from the
removal of lysozyme molecules, that is, lysozyme molecules act as
a template at this temperature. This suggestion is also supported
by SEM and TEM observation of powder samples calcined at
500 uC, showing that porous shell structures without through-holes
were formed. The nitrogen sorption isotherms of L-SHHs calcined
at 500 uC exhibit a diagnostic type IV isotherm with very little
hysteresis consistent with these TEM observations. These results
also prove that the lysozyme molecules are dispersed one by one
within L-SHHs without aggregation. The extinction of the 3 nm
diameter mesopores and the appearance of through-holes on
increasing the calcination temperature from 600 to 700 uC imply
the reconstruction of the silica network after the removal of the
lysozyme. Bicontinuous structures observed among the CHS by
SEM suggest that these morphologies are indicative of the
restructuring from mesoporous structures to a cage-like structure
Notes and references
{ A typical synthesis of CHS was performed as follows. A 1 ml volume of
TEOS was added to 9 ml of lysozyme solution (final concentration of
lysozyme: 2 mg ml21, 0.05 M glycine buffer, pH 9). Immediately the
mixture was sonicated for 15 min at RT. The resultant solutions were
dispensed onto a polystyrene plate and dried at 60 uC for 24 h, after which
a white powder was obtained (L-SHHs). The L-SHHs were then calcined
in air at 700 uC for 2 h. SEM observations of CHS and other silica samples
were carried out using a Hitachi S-800 instrument operated at 10 kV. The
nitrogen adsorption/desorption experiments were carried out using a
NOVA 3000 series instrument (Quantachrome Instruments) after drying
the sample powder at 200 uC for 2 h (except for as-synthesized L-SHHs:
60 uC for 24 h). The TGA data were recorded on a TG/DTA 300
instrument (SEIKO) at 10 uC min21 in an air atmosphere.
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