NANO

LETTERS

Urease Encapsulation in Nanoorganized 2001

Vol. 1, No. 3
Microshells 125-128

Yuri Lvov,*, Alexei A. Antipov, Arif Mamedov, Helmuth Mo1 hwald, and
Gleb B. Sukhorukov*,

Institute for Micromanufacturing, Louisiana Tech UniVersity, Ruston, Louisiana 71272,

Max Planck Institute of Colloids and Interfaces, Golm/Potsdam, D-14476, Germany,
and Department of Chemistry, Oklahoma State UniVersity, Stillwater, Oklahoma 74078

Received January 11, 2001 (Revised Manuscript Received February 15, 2001)

ABSTRACT
Stable hollow polyelectrolyte capsules were produced by means of the layer-by-layer assembling of poly(allylamine), PAH, and poly-
(styrenesulfonate), PSS, on melamine formaldehyde microcores followed by the core decomposition at low pH. These capsules are nonpermeable
for urease in water and become permeable in a water/ethanol mixture. The capsules were loaded with urease in water/ethanol mixture and
then resuspended in water. The urease molecules are kept in the capsule, whereas the small urea molecules rapidly diffuse through the
capsule wall providing a substrate for the biocatalytic reaction.

A thin film assembly by means of alternate adsorption of ability for dextran and albumin depends on pH: at low pH
oppositely charged linear polyions was introduced in the the capsule walls were open, and at pH higher than 8 they
nineties by Decher et al.1 The basis of this method involves were closed. By varying the pH in the capsule suspension
resaturation of polyion adsorption, resulting in the reversal in the presence of the macromolecules, the encapsulation was
of the terminal surface charge of the film after deposition of performed. An operation with opening-closing capsule walls
each layer. The method provides the possibility of designing composed of polyion multilayers is based on Rubners recent
ultrathin multilayer films with a precision better than one finding14 that varying solution pH can induce charge dis-
nanometer of defined molecular composition. The assembly balance in polycation-polyanion complexation in the mul-
process elaborated for planar solid supports was adapted for tilayer, resulting in opening of ca. 100-nm pores.
nano- and microtemplates (colloid particles with sizes of 0.1 In this work, as a part of our efforts to create unique and
to 5 microns, e.g., latex spheres, lipid tubules, microcrystals, complex colloids with tailored enzymatic activity, we report
biological cells, and other colloids).2-12 In this process, a the assembly of polyion capsules loaded with urease. In
polycation solution is added to the suspension of colloid traditional bioreactors, urease was immobilized by covalent
particles, and after adsorption saturation, the particles are bonding or with acrylamide gel on different substrates (glass
separated from free polycations in solution. Then, a polyanion beads or glass wool, nylon netting, nitrocellulose).15-20
layer is deposited. In the same manner, one can deposit any Nanocomposites containing urease multilayers on 470-nm
number of polyion layers on the shell. Recently, a multifil- latex were recently prepared.21
tration procedure for separation of modified particles from
We encapsulated urease in microshells assembled via
unreacted polyions was introduced,8 which allowed produc-
layer-by-layer assembly of linear polycations and polyanions
ing larger amounts of shelled particles, as compared with
on a 5-m diameter template. Hollow polyelectrolyte cap-
an earlier procedure based on separation of colloids and
sules were fabricated at pH 6.5 by alternate adsorption of
polyions by centrifugation.2-5 After the shells are formed,
one can dissolve the core particles to obtain empty capsules four bilayers of sodium poly(styrenesulfonate) (PSS) (Ald-
with a layer thickness tuned in the range from 5 to 50 nm rich, Mw 70,000)/poly(allylamine) hydrochloride (PAH)
and with needed composition.2-3,8 The permeability proper- (Aldrich, Mw 50,000), onto 5-m diameter melamine
ties of polyion capsules can be varied by shell composition formaldehyde (MF) particles (Microparticles Gmbh, Berlin),
and layer number.9,13 As shown in ref 12, the wall perme- using sequential adsorption with the filtration method.4,22 The
latex templates were dissolved, and the obtained microcap-
* Corresponding authors. E-mail: [email protected], gleb@mpikg-
sules were loaded with urease.
golm.mpg.de.
Louisiana Tech University.
A confocal fluorescence image23 in Figure 1 (left) il-
Max Planck Institute of Colloids and Interfaces. lustrates that FITC-labeled urease with concentration 10 mg/
Oklahoma State University. mL is excluded by the polyelectrolyte shells. The interior of
10.1021/nl0100015 CCC: $20.00 2001 American Chemical Society
Published on Web 03/14/2001
 as follows: The macromolecules were first exposed to
polyion capsules in a water/ethanol mixture, then ethanol
was removed after centrifugation and the capsules were
resuspended in water.24 After that, the polyion capsule walls
were closed and the urease was captured inside, as illustrated
in Figure 1 (right). The interior of the capsule is bright and
constant over time, and there is no fluorescence signal from
the solution. Thus, urease filled the capsules. The images
did not change with time, indicating that urease is preserved
inside the capsules. The fluorescence near the inner capsule
surface is brighter than in the center of the capsule. This is
due to adsorption of proteins onto adhesive polyion multi-
Figure 1. Permeation and encapsulation of urease-FITC into layers, as was observed earlier for dextran-loaded capsules.12-13
polyion multilayer capsules. Left, in water; middle, in water/ethanol The mechanism of reversible permeability changes in the
mixture 1:1; right, the capsule with encapsulated urease again in polyion multilayers is not yet understood. Probably, it is
the water. Top, scheme; bottom, confocal fluorescence images of
the capsules.
related to segregation of the polyion network in water/ethanol
media. Such segregation might lead to defects in the shell,
and pores might be open big enough to pass 5-nm diameter
the capsules remains dark and outside the capsule the
urease globules through the wall. Returning capsules into
background containing FITC-urease is fluorescent. Hollow
pure water causes a relaxation of the polyion walls to a closed
polyelectrolyte capsules are not permeable for compounds
with large molecular weight, such as proteins.9,12 This structure.
observation indicates the closed state of the capsules. Figure Figure 2 a,b gives TEM images (JEOL-2000 FX instru-
1 (center) shows a confocal fluorescence image of labeled ment, 200 kV) of empty and loaded capsules. An empty
urease with capsules after addition of ethanol (1:1 water capsule looks like a folded thin shell, but a loaded capsule
ethanol mixture). In this case, the fluorescence coming from looks like a filled balloon, similar to the optical image (Figure
the interior of the capsule is the same as the outside 1). We succeeded in filling the enzyme inside the capsule,
background fluorescence. This points at penetration of protein but we cannot quantify the enzyme content. To estimate the
into the capsules and indicates an open state of the capsule weight of encapsulated urease, we weighed an equal number
wall. of empty and loaded capsules with a quartz crystal microbal-
The transition between the open and closed state of the ance, QCM.25 QCM frequency shifts are proportional to the
polyelectrolyte multilayer capsules introduced by adding mass attached to an electrode (F), and the ratio Floaded/
ethanol in aqueous solution is reversible. When the capsules Fempty is proportional to the mass ratio of loaded and empty
are transferred into water, the polyion shells become closed capsules dried on the QCM electrodes. This ratio was found
for urease molecules. The reversible reorganization of to be ca. 1.1, indicating that the loaded capsule is 10%
polyions in water and water/ethanol mixture resulting in a heavier than the empty one. If we assume a similar hydration
permeability change was utilized for urease encapsulation of the dried shells, we can estimate the mass of the urease

Figure 2. TEM images: empty (a) and urease loaded (b) microcapsules, magnification: 300 K.
126 Nano Lett., Vol. 1, No. 3, 2001
 the bioactivity for urease immobilized with polycations on
latex dropped to 25% as compared with free enzyme.21 The
urease activity inside the capsules was also stable as
compared with free urease: after 5 days storage at 7 C,
encapsulated urease completely preserved its activity while
free urease kept at the same conditions in aqueous solution
lost 45% activity. The polyion shell protects encapsulated
enzymes from proteases and microbes, as was indicated in
ref 26.
The concept of enzyme encapsulation in microshells by
opening and closing pores can be applied to fabrication of
enzymatic micro- and nanoreactors. Selective wall perme-
ability allows for substrates and reaction products to diffuse
Figure 3. Absorbance at 588 nm by 2.9 mL of enzymatic activity freely through capsule walls, whereas the encapsulated
assay solution after addition of 0.1 mL of urease-loaded micro- enzymes are kept in the capsules. This technology provides
capsule solution and the same test for addition of 0.05 mL of 0.02 also a possibility to encapsulate several proteins at the same
mg/mL free urease. time in the capsules to catalyze sequential enzymatic
reactions. The loaded enzymes might be released out of the
kept inside the capsule. These estimations are approximate
capsule after certain pH or salt treatment, which could find
and are based on the assumption that the wall thickness is
some application in drug delivery and sustained release
much smaller that the diameter of the shell. The mass of the
systems.
capsule is m ) 4r2hF ) 1.38 10-12 g, where r ) 2.5
10-4 cm is the capsule radius, h ) 16 10-7 cm is the wall
Acknowledgment. The authors thank Prof. Bill Elmore
thickness,1-3 and the polyion density r ) 1.1 g/cm3. The
(LaTech) and Dr. Olga Tiourina for valuable discussion and
mass of the urease loaded in one capsule of the volume of
comments and Prof. Nicholas Kotov (Oklahoma State
6.3 10-11 cm3 is ca. 1.4 10-13 g, which corresponds to
University) for TEM images of the capsules. Y.L. acknowl-
170,000 urease molecules or the concentration of 2.1 mg/
edges the donors of the Petroleum Research Fund, admin-
mL. This urease concentration is close to the initial concen-
istered by American Chemical Society, and the Max-Planck
tration used for the loading from water/ethanol solution.
Society for support of this research.
A colorimetric assay based on the hydrolysis of urea was
used for the activity control of free and immobilized urease,
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Nano Lett., Vol. 1, No. 3, 2001 127

 (21) Lvov, Y.; Caruso, F. Anal. Chem. 2001, in press. dried. By frequency shift, we calculated the mass of capsules in the
(22) Capsule preparation. PSS and PAH were used in water solutions at solutions. The long-term stability (several hours) of the quartz
a concentration of 2 mg/mL with 0.5 M NaCl. For this we added 10 resonator frequency was within (2 Hz. The resonators used were
mL of aqueous PSS solution to 50 mL solution of 10 wt% MF- coated with evaporated gold electrodes (0.16 cm2) on both faces,
latex, allowed 20 min for adsorption saturation, removed unreacted and their resonance frequency was 9 MHz (AT-cut). For the QCM
PSS by filtration, and washed the modified latex with pure water. electrodes used in this work, the following relationships between the
At the second stage, aqueous PAH was added, adsorbed, and excess frequency shift (F, Hz) and polyion layer mass (M, g) is valid: M
polycation was filtered out. A stepwise application of this procedure ) -0.87 10-9F.27-28
resulted in formation of (PAH/PSS)4 shells. Afterwards, we decreased
(26) Onda, M.; Ariga, K.; Kunitake, T. J. Ferment. Bioengin. 1999, 87,
the solution pH to pH ) 1, latex-cores were dissolved, and empty
69.
shells were washed with water to remove MF-residues. Thus, the
hollow capsules were produced. (27) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc.
(23) Confocal laser scanning microscopy. Confocal micrographs were 1995, 117, 6117.
taken with Leica TCS SP, equipped with a 100 oil immersion (28) Sauerbrey, G. Z. Physik 1959, 155, 206.
objective. The investigated capsule suspension was placed between (29) Urease activity assay. A colorimetric assay based on the hydrolysis
glass slide and cover slip glued at the edges. Excitation wavelength of urea was used for the activity control of free and immobilized
488 nm was chosen accordingly to fluorescein labels. urease, as monitored by the pH-sensitive dye bromcresol purple. A
(24) To encapsulate urease, one part of empty capsule suspension was test solution containing 25 mM urea, 0.015 mM bromcresol, and 0.2
mixed with 2 parts of 96% ethanol, and 1 part of 10 mg/mL urease mM EDTA was adjusted to pH 5.8. This solution was placed in a
solution (U-4002, Sigma) was added. After a 10 min incubation UV cell and stirred. A known amount of the urease-loaded capsules
period, the capsules were centrifuged and washed with water to was then added to this solution, and the kinetics was monitored by
remove ethanol. following the UV absorption at 588 nm. The increment of the
(25) To control the weight of empty and loaded capsules, the quartz crystal absorbance with time was used to characterize the urease activity in
microbalance technique (QCM, USI-System, Japan) was used.27 In the sample.18,19
the measurements, 5 L of empty shells or loaded capsules with equal
particle concentrations were dropped onto the QCM electrode and NL0100015

128 Nano Lett., Vol. 1, No. 3, 2001