Langmuir 2000, 16, 1485-1488 1485

Enzyme Encapsulation in Layer-by-Layer Engineered

Polymer Multilayer Capsules
Frank Caruso,*,† Dieter Trau,‡ Helmuth Möhwald,† and Reinhard Renneberg‡
Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany, and Department
of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay,
Kowloon, SAR Hong Kong, China
Received August 27, 1999. In Final Form: November 23, 1999

We report on the encapsulation of enzyme (catalase) by the controlled polymer multilayer coating of
biocrystals, achieved by the sequential adsorption of oppositely charged polyelectrolytes onto enzyme
crystal templates. An extremely high enzyme loading in each polymer capsule is obtained, and the activity
of the encapsulated enzyme is preserved. The polymer-encapsulated enzyme is stable against protease
degradation: The polymer-coated enzyme retains 100% of its activity after incubation for 100 min with
protease, whereas uncoated, solubilized catalase loses more than 90% of its initial activity within 100 min
under the same conditions. This simple, general, and versatile approach can potentially be applied for the
encapsulation of various crystallized substances for catalysis and drug delivery applications.

Encapsulation technologies are utilized in medicine,
pharmaceutics, agriculture, and the cosmetic industries
for the development of controlled-release delivery
systems.1-3 Carrier systems of colloidal dimensions, such
as liposomes, polymeric particles, and microemulsion
droplets, are used for the sustained release of drugs,
pesticides, fragrances, and other substances.3,4 Although
such delivery systems are widely employed, problems
associated with their stability and permeability are often
encountered, thereby limiting their general application.
Here we present a novel and general approach to using
biocrystals as templates for the controlled encapsulation
of biomolecules (enzyme) by polyelectrolyte multilayers,
as illustrated in Figure 1. The method takes advantage
of the fact that the enzyme is a crystalline suspension in
water at pH 5-65 and therefore can be treated as a colloidal
particle. We earlier demonstrated that charged polymer
particles can be successfully employed as colloidal cores
for the assembly of alternating charged polyelectrolyte
multilayers.6-9 Removal of the colloidal core via its
decomposition and penetration of its constituent oligomers
through the polyelectrolyte walls resulted in hollow
polyelectrolyte capsules.7,8 A similar strategy, where
nanoparticles were deposited in alternation with poly-
electrolyte, and the core subsequently removed, was
recently also employed to produce inorganic and inorganic-
hybrid hollow spheres.10 Enzyme crystal templating,
however, presents several challenges that do not apply
when templating, for example, latex particles. First, the
crystals are formed under delicate solution conditions;
hence suitable conditions that facilitate polymer multi-
layer deposition on the crystal surface and do not destroy
the enzyme crystal morphology (i.e. to avoid its solubi-
lization) need to be found. Second, the permeability of the
polymer capsule walls must be such that it permits
encapsulation of the enzyme. In addition, since the primary
usefulness of enzymes is their biological function, their
activity should be preserved when encapsulated. Here we
describe a method for enzyme encapsulation that satisfies
all of these criteria.
In our enzyme crystal templating procedure,11 the
catalase crystals were separated from already solublilized
protein by washing and centrifuging several times with
1 M potassium acetate buffer of pH 5 at 4 °C. Chilled
solutions were used to avoid significant solubilization of
the enzyme crystals. A tapping mode atomic force mi-
croscopy image12 of a catalase crystal, approximately 8 ×
12 µm in size, is shown in Figure 2A. The catalase crystals
exhibit a positive surface charge in water at pH 5 (zeta
(ζ)-potential ) +20 mV, catalase isoelectric point ) 5.8),
as determined by electrophoretic mobility measure-
ments.13 This positive charge on the surface of the crystals

* To whom correspondence should be addressed. Fax: +49 331
567 9202. E-mail: frank.caruso@mpikg-golm.mpg.de.
† Max Planck Institute of Colloids and Interfaces.
‡ The Hong Kong University of Science and Technology.
(1) Langer, R. Nature 1998, 392, 5-10.
(2) Park, K. Controlled Drug Delivery: Challenges and Strategies;
American Chemical Society: Washington, DC, 1997.
(3) Kreuter, J. Colloidal Drug Delivery Systems; Marcel Dekker: New
York, 1994.
(4) Yokoyama, M.; Okano, T. Adv. Drug Delivery Rev. 1996, 21, 77-
80.
(5) Biochemica Information; Boehringer: Mannheim Germany, 1987;
pp 15-16.
(6) Caruso, F.; Donath, E.; Möhwald, H. J. Phys. Chem. B 1998, 102,
2011-2016.
(7) Sukhorukov, G. B.; Donath, E.; Davis, S. A.; Lichtenfeld, H.;
Caruso, F.; Popov, V. I.; Möhwald, H. Polym. Adv. Technol. 1998, 9,
759-767.
(8) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Möhwald,
H. Angew. Chem. 1998, 110, 2324-2327; Angew. Chem., Int. Ed. 1998,
37, 2201-2205.
(9) Caruso, F.; Lichtenfeld, H.; Donath, E.; Möhwald, H. Macromol-
ecules 1999, 32, 2317-2328.
(10) Caruso, F.; Caruso, R. A.; Möhwald, H. Science 1998, 282, 1111-
1114.
(11) The enzyme crystals were encapsulated as follows: The catalase
crystals (Sigma, C-100) were washed twice with a chilled (4 °C) solution
of 1 M potassium acetate at pH 5 (buffer), with intermittent centrifuga-
tion steps (500 g, 4 min, 4 °C) to remove the supernatant. The polymer
layers were then assembled onto the enzyme crystals by the sequential
deposition of poly(sodium 4-styrenesulfonate) (PSS), Mw 70 000, and
poly(allylamine hydrochloride) (PAH), Mw 8000-11000, both obtained
from Aldrich. PSS was dialyzed against Milli-Q water (Mw cutoff 14 000)
and lyophilized before use. The first layer was deposited by adding a
0.5 mL aliquot of a 5 mg mL-1 aqueous PSS solution (containing 1 M
potassium acetate, pH 5, 4 °C) to 0.2 mL of the crystal suspension,
occasionally shaking the suspension, and allowing 25 min for adsorption.
The excess polyelectrolyte was removed by three repeated centrifugation
(500g, 4 min, 4 oC)/chilled buffer wash/redispersion cycles. The next
layer, PAH, was deposited from a 5 mg mL-1 solution containing 1 M
potassium acetate (pH 5 at 4 °C) using the same procedure and
conditions. Subsequent alternating PSS and PAH layers were deposited
in identical fashion until the desired number of polymer multilayers
was achieved.
(12) AFM images were obtained with a Digital Instruments Nano-
scope IIIa AFM in tapping mode (TM) on samples deposited onto cleaned
glass slides and air-dried.

10.1021/la991161n CCC: $19.00 © 2000 American Chemical Society

Published on Web 01/29/2000
1486 Langmuir, Vol. 16, No. 4, 2000 Letters

Figure 1. Scheme showing the process used to encapsulate enzymes by using biocrystals as templates for the deposition of polymer
multilayers, subsequent enzyme solubilization and release, and the formation of hollow polymer capsules. (1, 2) Polyelectrolyte
layers are deposited stepwise onto the crystals by making use of the surface charge reversal that occurs upon adsorption of each
layer. Each polyelectrolyte layer deposited bears an opposite charge to that already adsorbed. Excess, unadsorbed polyelectrolyte
is removed by repeated centrifugation/wash redispersion cycles before the next layer is deposited. (3) Solubilization of the enzyme
inside the polymer capsule by exposure to solutions of pH > 6 or acidic solution (pH < 4) results in a morphology change of the
polymer capsule. (4) Release of the enzyme by rupturing the polymer capsule, achieved by exposure to solutions of pH > 11. (5)
Exposure of the encapsulated enzyme to an oxidizing solution results in decomposition of the enzyme which then is expelled from
the interior through the polymer walls, leaving behind hollow polymer capsules that originally encapsulate the enzyme.

in principle makes them suitably charged templates for
the deposition of polyelectrolyte layers. Next, the first
polyelectrolyte layer, poly(styrenesulfonate) (PSS), which
is negatively charged, was added to the crystal suspension
and allowed to self-assemble onto the crystal (Figure 1).
Removal of the excess, unadsorbed polyelectrolyte was
achieved by centrifugation of the crystal suspension,
removing the supernatant, and repeated washing with a
chilled potassium acetate solution. At this stage the
enzyme crystal surface charge was reversed (ζ-potential
of -30 mV), indicating the successful adsorption of PSS.
Repeated sequential deposition of poly(allylamine hydro-
chloride) (PAH) and PSS produced crystals with alternat-
ing positive (+20 mV) and negative (-30 mV) ζ-potentials,
respectively. These data clearly demonstrate a reversal
of the surface charge, which is characteristic of polyelec-
trolyte multilayer growth on colloidal templates.6-10
To verify that the enzyme crystals could be successfully
coated with polyelectrolyte multilayers, a fluorescently
labeled polyelectrolyte (fluorescein isothiocyanate (FITC)-
PAH) was substituted for each alternate polyelectrolyte
(polycation) in the buildup process. The fluorescence, as
seen under a fluorescence microscope, originates from the
surface of the polymer multilayer-coated enzyme crystals
(Figure 2B). The fluorescence signal was found to increase
systematically with an increasing number of fluorescent
layers (data not shown). No noticeable changes in size or
shape of the crystals were observed with the polyelectrolyte
multilayer shell coating. Further, the polymer-coated
enzymes could be stored for at least 30 days at 4 °C without
any noticeable change in morphology. These results show
that enzyme crystals can be encapsulated within a polymer
multilayer capsule via the stepwise, regular assembly of
oppositely charged polyelectrolytes and that the crystal
size and shape are retained upon their coating.
(13) Electrophoretic mobilities of the uncoated and polymer multi-
layer-coated enzyme crystals were measured with a Malvern Zetasizer
4 as described elsewhere.6-9 The mobility u was converted into a
ζ-potential using the Smoluchowski relation ζ ) uη/, where η and  are
the viscosity and permittivity of the solution, respectively.
Exposure of the polymer multilayer-encapsulated en-
zyme crystals to a solution of pH 2 resulted in the polymer
capsules assuming a more spherical shape (Figure 3A).
The morphology changes of the polymer capsules at pH
2 are attributed to solubilization of the enzyme crystal,
which occurs because the polyelectrolyte multilayers are
permeable to small mobile molecules (e.g., ions and water)
in the solution phase. (Previous fluorescence spectroscopy
studies have shown that similar polymer multilayers
assembled on solid core colloidal particles are permeable
to small polar molecules of diameter 1-2 nm.6,9) The more
spherical shape is most likely due to the osmotic pressure
buildup in the polymer capsules, caused by the high
concentration of dissolved enzyme. The capsules did not
rupture solely as a result of enzyme solubilization,
indicating their high stability. Similar morphology changes
were observed for other coated crystals. Catalase solu-
bilization readily occurred at pH < 4 and pH > 6.
Evidence that the enzyme was encapsulated within the
polymer capsules was obtained by deliberately rupturing
the polymer capsules. The polymer multilayer capsules
were found to partially rupture upon exposure to alkaline
solutions of pH > 11.14 An optical micrograph of capsule
rupture and enzyme release is displayed in Figure 3B.
Soon after capsule wall rupture, solubilized enzyme was
expelled from the interior of the capsule. Video microscopy
verified that the solubilized catalase was expelled upon
exposure to solutions of pH > 11 as a result of capsule
breakage. Capsule rupture and the associated expulsion
of the enzyme are caused by the combination of osmotic
pressure increase in the interior of the polymer capsules
(14) Several drops of the polymer multilayer-coated crystal solution
were placed on a cleaned glass slide and most of the water was allowed
to evaporate. An alkaline solution of pH > 11 was then pipetted onto
an area on the slide near that which contained the encapsulated enzyme
crystals. The alkaline solution was then allowed to make contact with
the coated enzymes, and the polymer capsule rupture and enzyme release
were monitored using optical and video microscopy. Rupture occurred
immediately upon contact with the alkaline solution front. It should be
noted that this release process may not be suitable for certain
applications. To this end, we are currently employing a range of other
polymers in order to effect enzyme release.
Letters
Figure 2. Images of uncoated and polymer multilayer-coated
catalase crystals. (A) AFM image of an uncoated crystal showing
its shape and micrometer dimensions. (B) Fluorescence optical
micrographs of catalase crystals coated with eight [(PSS/FITC-
PAH)4] polyelectrolyte multilayers, showing the applicability
of the coating process to enzyme crystal templates. The
fluorescently labeled (fluorescein isothiocyanate, FITC) poly-
cation, FITC-PAH, is deposited in alternation with the
polyanion, PSS.
and most probably a weakening of the electrostatic
interactions between the charged moieties of the polymers
due to the basic solution (pKa (PAH) is about 10).
Figure 4 shows a representative TEM image of an air-
dried polymer capsule,15 which was obtained after exposing
the polymer-coated enzyme crystals to an oxidizing
solution (deproteinizer).16 The enzyme was decomposed
by the deproteinizing treatment, allowing the expulsion
of its fragment constituents from the interior by permeat-
ing the polymer capsule walls. The drying process
(evaporation of the aqueous content by air-drying) induces
a number of folds and creases in the polymer capsules.
The capsules are also flattened and some spreading occurs;
the diameters of the dried polymer capsules are ap-
(15) Samples for transmission electron microscopy (TEM, Philips
CM12 microscope operating at 120 kV) were prepared by deposition of
aqueous solutions of the hollow polymer capsules upon a carbon-coated
copper grid, allowing them to air-dry for 1 min, and then blotting off
the extra solution.
(16) Hollow polymer capsules were obtained by exposing the polymer
multilayer-coated capsules to a deproteinizer solution (Medical instru-
ments; active constituent is sodium hypochlorite) for 15 min and washing
three times with 100 mM sodium chloride solution. Although the pH
of the deproteinizer solution is approximately 12, no capsule rupture
was observed. This is most likely due to the rapid decomposition of the
enzyme, hence avoiding any significant increase in osmotic pressure
buildup in the capsules.
Langmuir, Vol. 16, No. 4, 2000 1487
Figure 3. Representative optical micrographs of the polymer
capsules encapsulating solubilized enzyme. (A) A polymer
multilayer capsule containing solubilized enzyme in its interior.
The close to spherical shape is assumed upon solubilization of
the enzyme as a result of exposure to a solution of pH 2.
Solubilization also occurs upon exposure to solutions of pH <
4 or pH > 6. (B) Rupturing of a polymer multilayer capsule
causes the release of the entrapped, solubilized enzyme.
Rupturing is achieved by subjecting the polymer capsules to
alkaline solutions of pH > 11.
proximately 20 µm. The texture of the capsules is
characteristic of the polyelectrolyte film,8 although some
residual (undecomposed) catalase can be seen. From AFM
examination of air-dried polymer capsules (data not
shown), it can be deduced from the lowest height dimension
(35 nm), which is equivalent to twice the polymer capsule
wall thickness, that the average thickness per polyelec-
trolyte layer is approximately 2 nm (the enzyme crystal
templates were coated with eight layers). This value is
consistent with data obtained for multilayers on solid core
particles.7-9
The activity of the encapsulated catalase and the
stability imparted by the polymer multilayer coating with
respect to proteolysis were examined. The catalase activity
was measured after its solubilization and release from
the polymer capsules. This was achieved by following its
reaction with hydrogen peroxide (substrate) to produce
water and oxygen.5,17 A recovered specific activity of 97%
was obtained, compared with 100% for the uncoated
catalase. This shows that the polymer multilayer coating
of the catalase crystals proceeds without causing any
significant loss of enzyme activity. The results of the
1488 Langmuir, Vol. 16, No. 4, 2000
Figure 4. TEM image of an air-dried hollow polymer capsule
comprising eight [(PSS/PAH)4] polyelectrolyte layers, obtained
after decomposition of the encapsulated enzyme. The polymer
capsule spreads out on the carbon surface on which it is dried,
and folds and creases can be seen. Some undecomposed enzyme
can still be seen in the interior of the capsule.
proteolysis experiments18 are shown in Figure 5. Solu-
bilized, uncoated catalase (curves d and e) was inactivated
by protease to more than 90% during an incubation time
of 100 min. In contrast, no measurable loss in enzyme
activity was observed for the polymer-encapsulated (solu-
bilized) catalase within 100 min under the same conditions
(curves b and c). (The catalase was solubilized and retained
within the polymer multilayer capsule at the experimental
pH of 7.) Further experiments showed that the activity
of the encapsulated enzyme was fully retained after 24 h
of exposure to protease. These results clearly demonstrate
that a thin polymer coating of four layers (thickness of ca.
8 nm) is sufficient to prevent proteolysis of the polymer-
encapsulated catalase. This finding is consistent with the
observation that proteins of diameter greater than ap-
(17) The enzyme activity was determined by first releasing the enzyme
from the polymer capsules by exposure to ultrasound for 5 min at 20
°C in 1 M phosphate buffer at pH 7 (this caused rupturing of the polymer
walls) and using a standard enzyme activity assay;5 that is, by measuring
the decomposition of the substrate hydrogen peroxide (H2O2) at 240
nm. The concentration of the catalase was determined spectrophoto-
metrically at 280 and 405 nm (soret band). The specific activity was
obtained by dividing the measured activity by the enzyme concentration.
The specific activity of the uncoated catalase was measured in the same
way and used for comparison. See also, for example: Tijsen, P. Practice
and Theory of Enzyme Immunoassays, 8th Impression, Elsevier:
Amsterdam, 1993; pp 202-203.15.
(18) 10 mg of protease (Streptomyces griseus; Sigma P6911; activity
) 5.2 U/mg solid) was dissolved in 500 µL of phosphate buffered saline
(PBS, pH 7.0) (100 U/ml) and used as the stock solution. Samples (200
µL) of the polyelectrolyte-coated catalase and solublilized catalase were
separately incubated in a water bath at 37 °C with either 30 µL of the
stock protease solution or 30 µL of PBS (control experiment). The starting
activity of the catalase for all samples was approximately 12 000 U/mL
catalase. The starting activities were normalized to 100%. The final
protease activity in the sample was 13 U/mL. Proteolysis of the catalase
was determined by measuring the decrease in the catalase enzyme
activity. The catalase activity measurements where carried out according
to the previously described method (i.e., by spectrophotometric detection
(240 nm) of the decomposition of hydrogen peroxide, see ref 17). Samples
(5 µL) were diluted and used for the activity measurements.
Letters
Figure 5. Stability of (a, d, e) solution-solubilized catalase
and (b, c) polymer-multilayer encapsulated (solubilized) catalase
with respect to proteolysis: (a) solution-solubilized catalase
crystals, no protease incubation (control); (b) [(PSS/PAH)2]-
coated (four layers) catalase, protease incubation; (c) [(PSS/
PAH)4]-coated (eight layers) catalase, protease incubation; (d)
and (e) repeat experiments for solubilized catalase, protease
incubation. Proteolysis of the catalase was determined by
measuring the decrease in the catalase enzyme activity (see ref
18 for details).
proximately 5 nm do not penetrate polyelectrolyte mul-
tilayer films.19
Important advantages of the enzyme crystal templating
procedure presented are its versatility and generality: The
thickness and composition of the polymer walls can be
controlled by varying the number of polymer deposition
cycles, thus potentially providing a straightforward and
simple means to vary the permeability of the polymer
capsules. It is expected that this process can be extended
to encapsulate other biological materials in crystalline
(or amorphous) form, e.g. peptides, antibodies, or enzyme
catalysts in order to perform chemical reactions. Fur-
thermore, the volume in the polymer capsule consists of
an active catalyst (catalase). The layer-by-layer engineer-
ing of polymer multilayer capsules on biocrystal templates
also represents a novel way to prepare immobilized
catalysts with enhanced stability and activity, with the
polymer multilayer coating providing a protective barrier
for the enzyme in environments where enzyme-degrading
substances (e.g., macromolecules, microorganisms, etc.)
may be present. Overall, the strategy employed in this
work provides a new route to produce tailored and
optimized systems for numerous applications in biotech-
nology.
Our current work is focusing on using various crystal
templates, pH swellable, thermally responsive, and bio-
compatible polymers as the capsule constituents, on
constructing microreactor systems for sequential enzy-
matic biocatalytic reactions, and on functionalizing the
outer polyelectrolyte layers for targeting.
Acknowledgment. This work was supported by The
German Federal Ministry of Education, Science, Research
and Technology (BMBF) and the Max Planck Society. We
thank C. Dürr for technical assistance and M. Giersig for
help with electron microscopy.
LA991161N
(19) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir
1997, 13, 3427-3433.