Biomaterials 32 (2011) 1967e1977

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Mucosal irritation potential of polyelectrolyte multilayer capsules

Liesbeth J. De Cock a, Joke Lenoir a, Stefaan De Koker b, Vincent Vermeersch c, Andrei G. Skirtach d,
Peter Dubruel c, Els Adriaens a, Chris Vervaet a, Jean Paul Remon a, Bruno G. De Geest a, *
a
Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
b
Department of Molecular Biomedical Research, Ghent University, Technologiepark Zwijnaarde 927, 9052 Zwijnaarde, Belgium
c
Polymer Chemistry and Biomaterials Research Group, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium
d
Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, Golm, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Polyelectrolyte multilayer capsules have recently gained interest as carriers for drug delivery. When
Received 15 October 2010 envisioning mucosal administration, one is focused with potential concerns such as tissue irritation and
Accepted 6 November 2010 tissue damage, induced by the carrier itself. In this paper we demonstrate the use of a slug-based (Arion
Available online 3 December 2010
lusitanicus) assay to evaluate the mucosal irritation potential of different types of polyelectrolytes, their
complexes and multilayer capsules. This assay allows to assess in a simple yet efficient way mucosal
Keywords:
tissue irritation without using large numbers of vertebrates such as mice, rabbits or non-human
Mucosa
primates. We found that although single polyelectrolyte components do induce tissue irritation, this
Microcapsule
Drug delivery
response is dramatically reduced upon complexation with an oppositely charged polyelectrolyte,
Heparin rendering fairly inert polyelectrolyte complexes. These findings put polyelectrolyte multilayer capsules
Biocompatibility further en route towards drug delivery applications.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Polyelectrolyte multilayer capsules (PMLC) are a new type of
carrier system which hold high potential for drug delivery and
tissue engineering as they are well suited to efficiently encapsulate
and deliver antigens, growth factors, nucleic acids, anticancer drugs
etc. [1e3]. These capsules are fabricated by layer-by-layer (LbL)
coating of a sacrificial template followed by dissolution of this
template, yielding hollow capsules. A very convenient method to
encapsulate proteins into PMLC was recently described by Volodkin
et al. by co-precipitating a protein of choice with calcium chloride
and sodium carbonate forming calcium carbonate (CaCO3) micro-
particles with the protein entrapped within its porous structure
[4e6]. Subsequently, the CaCO3 microparticles are LbL coated with
polyelectrolytes of alternating charge and in a last step the CaCO3
cores are dissolved by extraction of the Ca2þ ions with an aqueous
EDTA solution (Fig. 1). As the polyelectrolyte complex shell is semi-
permeable, water, ions and low molecular weight species can freely
diffuse in and out but proteins remain entrapped. The major assets
of this type of capsule are (1) the mild conditions used for protein
encapsulation avoiding organic solvents, reactive chemistries or
* Corresponding author. Tel.: þ32 9 264 81 89; fax: þ32 9 222 82 36.
E-mail address: Br.DeGeest@UGent.be (B.G. De Geest).
high shear forces and (2) the ability to tailor the capsules’ physico-
chemical and biochemical properties by choosing from a virtually
unlimited range of capsule components [7e12]. Only incubation of
in vitro cultured cells with extremely elevated concentrations of
PMLC induced toxicity [13,14].
Recently, a number of papers from our group and others have
demonstrated the potential of PMLC for vaccine delivery where
protein antigens are encapsulated within the PMLC and efficiently
delivered to antigen presenting cells in vitro as well as in vivo
[1,14e17]. Formulating antigens into microparticles has a number
of distinct advantages compared to the delivery of soluble antigen
including protection against degradation, enhanced uptake by
professional antigen presenting cells such as dendritic cells and the
ability to induce ‘cross-presentation’ of antigen to both CD4 and
CD8 T cells, inducing a broad humoral as well as cellular immune
response [18,19]. As alternative to the parenteral route where the
vaccine is administered through subcutaneous or intramuscular
injection there is an increased interest in mucosal vaccination for
a number of reasons such as (1) the avoidance of needles, (2) the
possibility to store and apply the vaccine as dry powder, avoiding
the cold chain and (3) most importantly, the ability to vaccinate at
that specific site where most infectious organisms enter the body,
i.e. at mucosal surfaces, inducing a protective mucosal immune
response in addition to a systemic response.

0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.11.012
1968 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977

Fig. 1. Schematic overview of the fabrication of protein-loaded polyelectrolyte capsules using CaCO3 microparticles as sacrificial core templates. (A) Co-precipitation of the protein
(orange) in CaCO3 particles by mixing solutions of CaCl2 and Na2CO3 in the presence of a protein of choice. (B) Layer-by-layer coating of CaCO3 particles (green ¼ polyanion,
brown ¼ polycation). (C) Hollow polyelectrolyte capsules filled with the protein obtained after dissolving the CaCO3 particles with EDTA.

However, in order to reach a clinical stage, it is important to have
extensive data on mucosal irritation since this delivery route
induces contact between the drug carrier and mucosal surfaces. In
the past, several tests using vertebrates such as mice, rabbits or
non-human primates were developed to test mucosal irritation
potency of different components [20e22]. Besides being often
laborious, such assays raise important ethical questions concerning
the large turnover of laboratory animals. By consequence there is
need for alternative tests involving lower species that allow an easy
and fast in vivo assay to investigate mucosal irritation. Recently,
Adriaens et al. introduced an assay using terrestrial slugs, namely
Arion lusitanicus as a nonvertabrate model organism to investigate
mucosal irritation of several pharmaceutical as well as health care
components in vivo [23,24]. What makes slugs interesting as a test
organism is the morphology of their body wall which is composed
of an outer single epithelium layer, containing epithelial and
mucous gland cells overlying connective tissue. Observing the
effect of test components after contact is easy due to the location of
the epithelium layer which is at the outside of the body wall. Under
normal circumstances, slugs produce a limited amount of mucus
for their locomotion and to prevent dehydration. When slugs are
exposed to an irritating component, they will secrete mucus as
a mechanism to protect their body wall. The higher the amount of
mucus produced by the slugs, the more irritating the substance is
for their body wall. Also the color of the produced mucus indicates
the degree of irritation. Normally the mucus produced by slugs is
colorless however contact with irritating substances evokes
secretion of slightly yellow to dark yellow colored mucus. Serious
damage of the single-layered epithelium and the underlying
connective tissue results in an increased release of proteins and

Fig. 2. Chemical structure of the polyelectrolytes used for the layer-by-layer coating of CaCO3 microparticles. (A) heparin, (B) poly-L-arginine, (C) PSS, (D) PDADMAC, and (E) DAR.
 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977
Table 1
Amount of polyelectrolytes available in 10 mg hollow (PSS/PDADMAC)2, (PSS/
DAR)2 and (hep/pARG)2 capsules.
(PSS/PDADMAC)2 capsules
PSS 4.8 mg
PDADMAC 5.0 mg
(PSS/DAR)2 capsules
PSS 3.8 mg
DAR 6.1 mg
(hep/pARG)2 capsules
hep 5.7 mg
pARG 4.3 mg
enzymes such as lactate dehydrogenase (LDH) and alkaline phos-
phatase (ALP). LDH is a cytosolic enzyme and will be first released
in case of tissue damage. ALP is a membrane-bound enzyme and
will be released in case of severe tissue damage since it is only
present in the underlying connective tissue.
In this paper we assess the mucosal irritation potency of PMLC
composed of different types of polyelectrolyte pairs as shown in
Fig. 2: (1) Heparin/poly-L-arginine (hep/pARG) as ‘biopolymer’ LbL
film, (2) polystyrene-4-sulfonate/poly(diallyldimethylammonium
chloride) (PSS/PDADMAC) as synthetic LbL film and (3) poly-
styrene-4-sulfonate/diazoresin (PSS/DAR) as covalently linked
synthetic LbL film. Hep/pARG and PDADMAC/PSS interact purely
through electrostatics while under influence of light the diazo
groups of DAR react with the sulfonate groups of PSS forming
a tough impermeable coating [25,26]. The single polyelectrolyte
components were evaluated and compared with PMLC and with
polyelectrolyte complexes obtained by simply mixing the poly-
electrolytes in solution. The degree of mucosal irritation was
analyzed by evaluation of mucus production, the color of secreted
mucus, release of proteins and enzymes, and slug mortality within
a period of 7 days after exposure. The presented methodology is not
only restricted to PMLC but could be widely applied to a large
variety of nano- and micro-scale drug delivery systems that are
currently under investigation.
2. Materials and methods
2.1. Materials
Branched poly(ethyleneimine) (PEI; 22 kDa), polystyrene-4-sulfonate (PSS;
70 kDa), poly(diallyldimethylammonium chloride) (PDADMAC; 100e200 kDa) were
purchased from Sigma. Heparin (hep) was purchased from Diosynth biotechnology.
Diazoresin (DAR) was purchased from Livingston Associates, P.C. Poly-L-arginine
(pARG; 100 kDa) was a kind gift from Prof. Peter Dubruel (Polymer Chemistry and
Biomaterials Research Group; Ghent University). Drum-dried waxy maize (DDWM)
was obtained from National Starch.
Fig. 3. Schematic overview of the slug mucosal irritation test.
1969
2.2. UV/VIS analysis of layer-by-layer deposition
Quartz slides rinsed with piranha solution to render them more hydrophilic
were coated with PEI followed by deposition of layer-by-layer films based on
respectively PSS/DAR, PSS/PDADMAC and hep/pARG. Deposition of each poly-
electrolyte was performed by immersing the slide in a 1 mg/ml solution (containing
0.5 M NaCl) of the respective polyelectrolyte, followed by a washing step in water to
remove non-adsorbed polyelectrolyte. After each deposition step, the absorption
spectrum was recorded with a Shimadzu spectrophotometer.
2.3. Fabrication of polyelectrolyte capsules
Calcium carbonate microparticles (CaCO3) cores were fabricated by mixing equal
volumes of calcium chloride (CaCl2) and sodium carbonate (Na2CO3) solutions (1 M)
under vigorously stirring. The obtained CaCO3 core templates were subsequently
coated by incubating them alternately into a 1 mg/ml polyelectrolyte solution
containing 0.5 M NaCl during 10 min. After the incubation period, the capsules were
centrifuged and washed twice with water to remove non-adsorbed polyelectrolyte.
Two polyelectrolyte bilayers ((PSS/DAR)2, (PSS/PDADMAC)2 and (hep/pARG)2) were
deposited on the capsules and hollow capsules were obtained after extraction of the
CaCO3 core templates by addition of an aqueous 0.2 M EDTA solution.
2.4. Fabrication of polyelectrolyte complexes in solution
Complexes of PSS/DAR, PSS/PDADMAC and hep/pARG were made by mixing
equal amounts (w/w) of the respective oppositely charged (20 mg/ml containing
0.5 M NaCl) polyelectrolytes. The complexes were centrifuged and washed twice
with water to remove non-adsorbed polyelectrolyte.
2.5. Characterization of polyelectrolyte capsules and complexes
2.5.1. Scanning electron microscopy
Polyelectrolyte complexes, polyelectrolyte-coated CaCO3 microparticles and
hollow polyelectrolyte multilayer capsules obtained by dissolution of the CaCO3
cores were dried on a silicone wafer followed by sputtering with gold. SEM images
were recorded with a Quanta 200 FEG FEI scanning electron microscope operating at
an acceleration voltage of 5 kV.
2.5.2. Electrophoretic mobility
The electrophoretic mobility during multilayer build-up on the CaCO3 micro-
particles was measured using a Zetasizer Nano ZS (Malvern Instruments) equipped
with Dispersion Technology Software (DTS). The z-potential was calculated from the
electrophoretic mobility (m) using the Smoluchowski relation: z ¼ mh/3 where h and
3 are respectively the viscosity and permittivity of the solvent.
2.6. Elemental analysis
The composition of the respective polyelectrolyte complexes and capsules was
determined by elemental analysis (Vario EL, Elementar Analysensysteme).
2.7. Slug mucosal irritation test
Slugs were bred in the laboratory in plastic containers at 18e22  C and fed with
lettuce, cucumber, carrots and protein rich food. Slugs having a body weight of 3e6 g
were isolated 2 days before the start of the experiment, placed in a plastic box
containing a paper towel moistened with phosphate buffered saline and kept at
1970 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977

A 0.5 2.0 B 0.5

2.0

Absorbance [a.u.]

Absorbance [a.u.]
1.8
1.6 1.8 0.4
0.4 1.4
1.2 1.6 0.3
Absorbance [a.u.]

Absorbance [a.u.]
1.0 1.4
0.8 0.2
0.3 0.6 1.2
0.4 0.1
0.2 1.0
0.2 0.0 0.0
0 2 4 6 8 10 0.8 0 2 4 6 8 10
Number of bilayers 0.6 Number of bilayers
0.1 0.4
0.2
0.0 0.0
190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260
Wavelength [nm] Wavelength [nm]

Absorbance [a.u.] 0.5

C 1.0 0.4
0.3
0.8 0.2
Absorbance [a.u.]

0.1
0.6
0.0
0 2 4 6 8 10
0.4 Number of bilayers

0.2

0.0
200 250 300 350 400 450 500 550
Wavelength [nm]

Fig. 4. UV/VIS spectra of quartz slides coated with 10 bilayers of (A) hep/pARG, (B) PSS/PDADMAC and (C) PSS/DAR. The insets of graphs A, B and C show the absorbance of the
quartz slides as a function of the number of deposited bilayers at a wavelength of respectively 190 nm, 196 nm and 380 nm.

18e22  C. Only slugs without visible injuries on their foot surface and body wall
were used during the experiments.
The mucosal irritation potency was evaluated by placing the slugs on the test
substances in a Petri dish during 1 h. For each test substance 5 slugs were used. Slugs
were exposed to an amount of polyanion or polycation equal to the corresponding
amount of that polyelectrolyte present in 10 mg capsules as determined by
elemental analysis (Table 1). The amount of polyelectrolyte was supplemented with
drum-dried waxy maize (DDWM) to obtain a total mass of 10 mg. As a negative
control 10 mg DDWM was used and as a positive control 10 mg of a mix of DDWM
and sodium lauryl sulfate (DDWM/SLS 4/1) was used. In a next series of experiments,
total amounts of 10 mg lyophilized hollow capsules, respectively lyophilized poly-
electrolyte complexes were evaluated.
The mucus produced during the contact period of 1 h was measured by
weighing the Petri dishes with the test component before and after the contact
period and was expressed as % (w/w) of the initial body weight. After the contact
period with the test component, the slugs were placed on a new Petri dish con-
taining 1 ml of phosphate buffered saline (PBS; pH 7.4) during 1 h. After this first
1 h period on PBS, the slugs were transferred to another Petri dish containing 1 ml
PBS for the second 1 h period. The PBS samples of the two periods were collected
to quantify protein and enzyme release. A schematic overview of the slug mucosal
irritation test is shown in Fig. 3.
Quantification of released proteins was performed by the NanoOrangeÒProtein
Quantitation Kit (Molecular Probes) and is based on protein binding to the Nano-
Orange reagent at 95  C. The NanoOrange reagent as such is not fluorescent but
becomes fluorescent with an excitation at 470 nm and emission at 590 nm as soon as
it binds to proteins. Bovine serum albumin was used as a standard and fluorescence
was measured using a fluorometer (Wallac 1420 multilabel counter, PerkinElmer).
The protein concentration is expressed as mg/ml per g body weight.
Results of both mucus production and protein release (mean of the 2 samples)
were log-transformed because of the inhomogeneity of the variances and subjected
to a one-way ANOVA combined with a post-hoc Scheffé test.
2.8. Determination of enzyme activity
2.8.1. Alkaline phosphatase (ALP)
Alkaline phosphatase converts p-nitrophenyl phosphate into p-nitrophenol and
the enzyme activity was determined spectrophotometrically since p-nitrophenol
absorbs light at 405 nm in alkaline conditions. The amount of enzyme that catalyzes
the formation of 1 mmol/L of p-nitrophenol per minute under the assay conditions is
defined as one unit of ALP activity. The enzyme activity, determined with an enzyme
kit (ALP CP, Horiba CPX) was expressed as U/L per g body weight and measured on
a Cobas Mira Plus analyzer (ABX).
2.8.2. Lactate dehydrogenase (LDH)
Lactate dehydrogenase catalyzes the conversion of pyruvate into lactate with the
concomitant oxidation of NADH into NADþ and the rate of decrease in absorbance at
340 nm which is due to the oxidation of NADH is a measure for the LDH activity. The
amount of enzyme that catalyzes the formation of 1 mmol/L of NADþ per minute
under the assay conditions is defined as one unit of LDH activity. The enzyme
activity, determined with an enzyme kit (LDH CP, Horiba ABX) was expressed as U/L
per g body weight and measured on a Cobas Mira Plus analyzer (ABX).
2.9. Assessment of slug mortality
Slugs were placed on Petri dishes containing protein rich food and a small paper
towel moistened with PBS after exposure to test substances, and slug mortality was
determined at day 1, 2, 5 and 7.
2.10. Histological examination
After exposure, slugs were wrapped in aluminium foil and frozen in liquid
nitrogen. Cryosections of 10 mm were cut, stained with hematoxylin and eosin, and
examined by light microscopy.
 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977 1971

A 25 B 40
20 30
15
10 20
5 10
0
-5 0
-10 -10
-15 -20
-20
-25 -30
-30 -40
-35
0 1 2 3 4 hollow 0 1 2 3 4 hollow
Adsorption step Adsorption step

C 0
-5
-10
-15
-20
-25
-30
-35
-40
0 1 2 3 4 hollow
Adsorption step

Fig. 5. Zeta-potential profiles of the (A) (hep/pARG)2, (B) (PSS/PDADMAC)2 and (C) (PSS/DAR)2 multilayer build-up after each adsorption step on CaCO3 microparticles and after
adding EDTA (i.e. hollow capsules).

3. Results and discussion
3.1. Characterization of polyelectrolyte complexation on planar
surfaces, on sacrificial spherical templates and in solution
3.1.1. Formation of polyelectrolyte multilayer films on planar
surfaces
In first instance the LbL film forming ability of the different
polyelectrolyte pairs was studied. Therefore quartz slides were
chosen as planar substrates and after deposition of each polyanion/
polycation bilayer the absorbance was measured by means of
spectrophotometry. Ten bilayers heparin/pARG (Fig. 4A), PSS/
PDADMAC (Fig. 4B) and PSS/DAR (Fig. 4C) were deposited onto
a quartz slide and from the quasi linear increase of the absorbance
as a function of number of deposited bilayers, a successful multi-
layer build-up could be concluded.
3.1.2. Formation of polyelectrolyte films on sacrificial CaCO3
templates
After having demonstrated the possibility to form poly-
electrolyte multilayer films composed of hep/pARG, PSS/PDAD-
MAC and PSS/DAR on a flat substrate, spherical templates were
used to be coated with the appropriate multilayer films in a next
step. For this purpose CaCO3 microparticles were used since they
are, as mentioned earlier in the introduction of the work, espe-
cially well suited as sacrificial templates to form protein-loaded
PMLC. By means of measuring the electrophoretic mobility
(z-potential) after deposition of each polyelectrolyte layer, one is
able to monitor the multilayer build-up. Fig. 5 shows the
z-potential of the native CaCO3 microparticles, during LbL coating
and after dissolution of the CaCO3 cores with EDTA. Although
native CaCO3 particles exhibit a negative z-potential, the multi-
layer build-up was started with the polyanion. Due to their high
porosity, CaCO3 microparticles strongly adsorb polymers, regard-
less their charge and previous experiments on CaCO3 microparti-
cles (unpublished data) showed that inter-particle aggregation is
reduced when started with the polyanion.
Both (hep/pARG)2 (Fig. 5A) as well as (PSS/PDADMAC)2 (Fig. 5B)
coating resulted in an alternating behavior of the z-potential,
indicating that charge reversal takes place with each deposited
polyelectrolyte layer. Dissolving the CaCO3 cores by addition of
EDTA resulted in a decrease in z-potential from 11.9 mV to e19 mV
in case of (hep/pARG)2 capsules and from 31.3 mV to 14.3 mV in
case of (PSS/PDADMAC)2 capsules. This is likely due to reorgani-
zation of the polyelectrolyte within the capsule shell upon disso-
lution of the CaCO3 core and liberation of an excess of polyanion
that was adsorbed into the CaCO3 pores during the first deposition
step. Compared to (hep/pARG)2 and (PSS/PDADMAC)2 capsules, no
alternating z-potential profile was obtained for (PSS/DAR)2 capsules
(Fig. 5C). A z-potential of 23.1 mV was obtained after depositing
the first PSS layer and the z-potential remained negative after
depositing the following layers. This might be due to covalent
reaction between PSS and DAR, neutralizing the cationic diazo
moieties. Others did demonstrate an alternating profile during PSS/
DAR multilayer build-up when keeping the polymers in the dark
[25,26]. However, in our experiments we conducted the LbL build-
up in ambient light which is sufficient to induce coupling of both
polymers [27].
1972 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977

Fig. 6. Scanning electron microscopy images of (A) (hep/pARG)2 capsules, (B) (PSS/PDADMAC)2 capsules and (C) (PSS/DAR)2 capsules before (A1, B1, C1) and after (A2, B2, C2)
dissolution of the CaCO3 cores.

Scanning electron microscopy was further used to characterize
the PMLC. Fig. 6 shows micrographs before (A1, B1 and C1) and after
(A2, B2 and C2) dissolution of the CaCO3 core templates. The images
of collapsed spheres upon drying clearly demonstrate the forma-
tion of hollow capsules. A size distribution of roughly 3e5 mm is
observed and was confirmed by laser diffraction measurements.
3.1.3. Formation of polyelectrolyte complexes in solution
Polyelectrolyte complexes of respectively hep/pARG, PSS/PDAD-
MAC and PSS/DAR were made by mixing equal amounts (w/w) of the
oppositely charged polyelectrolytes in water. Fig. 7 shows scanning
electron microscopy images of the complexes dried onto a silicon
substrate. A similar granular morphology was observed for both the
complexes composed of hep/pARG (Fig. 7A) as well as the (hep/
pARG)2 coated CaCO3 microparticles (Fig. 6A1). By contrast, the
morphology of complexes composed of PSS/PDADMAC (Fig. 7B) and
PSS/DAR (Fig. 7C), both exhibiting large aggregated clumps, is
completely different compared to the surface of CaCO3 microparticles
coated with the appropriate polyelectrolyte pairs.
3.1.4. Composition of PMLC and polyelectrolyte complexes
For further studies the exact composition of the PMLC and
polyelectrolyte complexes is required. Therefore, elemental anal-
ysis was performed on hep/pARG, PSS/DAR and PSS/PDADMAC
capsules and complexes after extensive centrifugation and washing
steps to remove any excess of freely soluble polyelectrolytes.
Results of nitrogen, carbon, hydrogen and sulfur content are shown
in Table 2. Using the molecular weight of the repeating unit of each
polymer, the molar ratio of the polyanion to polycation was
calculated (Table 3).
 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977 1973

Fig. 7. Overview (1) and zoomed (2) scanning electron microscopy images of complexes composed of (A) hep/pARG, (B) PSS/PDADMAC and (C) PSS/DAR.

The molar ratio of heparin to poly-L-arginine in capsules is 0.349
indicating that a higher molar amount of poly-L-arginine is involved
in the layer-by-layer build-up on CaCO3 microparticles. This can be
easily understood since poly-L-arginine in solution has a tertiary
structure compared to heparin having a more linear structure. The
molar ratio of simple complexes composed of heparin and poly-L-
arginine is 0.351 which is remarkably similar to the value of PMLC.
PSS and PDADMAC exhibit a molar ratio of 0.755 for PMLC and
0.775 for simple polyelectrolyte complexes, also these values are
remarkably close. Apparently, hep/pARG and PSS/PDADMAC
interact in a similar fashion on a supported substrate as in solution.
Also in case of PSS/PDADMAC there is an excess of polycation, but
this is less pronounced than in case of hep/pARG.
In contrast to hep/pARG and PSS/PDADMAC, a large discrepancy
in the molar ratio of PSS/DAR capsules and simple complexes was
observed. PMLC have a PSS/DAR ratio of 1.118 while simple PSS/DAR
complexes have a ratio of 0.551. Thus, when depositing an LbL film
on CaCO3 microparticles, a higher molar amount of PSS is involved
while a higher molar amount of DAR is involved when forming
complexes in solution. This could be due to a higher availability of
sulfonate groups of PSS in solution whereby more DAR can interact
with PSS compared to the availability of sulfonate groups of
adsorbed PSS on CaCO3 microparticles.
3.2. Mucosal irritation potential of polyelectrolytes, polyelectrolyte
complexes and polyelectrolyte multilayer capsules
3.2.1. Mucus production
The different polyelectrolytes, their complexes and multilayer
capsules characterized in the sections above were tested for their
mucosal irritation potency by a slug mucosal irritation assay
developed by Adriaens et al. [23,24]. This assay is a reproducible
quantitative method which does not require complex test equip-
ment, sophisticated chemical analysis or a long experimental time.
Furthermore this assay did not require vertebrates such as mice,
rats and non-human primates resulting in a less laborious assay.
Slugs were exposed to the different samples in a dry powder
state obtained through lyophilization. The reason for this is that
dry powder is a common type of formulation for mucosal appli-
cation which often holds problems concerning mucosal irritation.
After 1 h exposure, we evaluated the mucus production by the
slugs, the color of the produced mucus, release of enzymes and
proteins, and slug mortality within a period of 7 days after
exposure. For PSS, 2 different amounts (denoted as PSS∼PSS/DAR and
PSS∼PSS/PDADMAC) were used according to the amount of PSS
present in respectively hollow (PSS/DAR)2 and (PSS/PDADMAC)2
capsules. The amount of a single polyelectrolyte component
available in 10 mg capsules could be calculated from the elemental
analysis results (Table 1). To classify our test substances for their
degree of irritation, the reactions of treated slugs were compared
to the reactions of slugs exposed to DDWM as negative and
DDWM/SLS (4/1) as positive control since previous research
revealed the non-irritating respectively irritating properties of
these substances [23,28].
Table 4 summarizes the results of total mucus production
(expressed as % (w/w) of the initial body weight) during exposure
to the test substances. The negative control evoked a minimal
secretion of colorless mucus which was on average 0.7% of the body
weight while the positive control evoked a high mucus secretion
(4.7% of the body weight) which had a yellow color.

Table 2 Table 3
Elemental analysis of hollow (hep/pARG)2, (PSS/DAR)2 and (PSS/PDADMAC)2 Molar ratio of polyanion to polycation in hollow capsules and complexes
capsules and the corresponding complexes. composed of hep/pARG, PSS/DAR and PSS/PDADMAC.

N(%) C(%) H(%) S(%) Molar ratio

Hollow (hep/pARG)2 capsules 14.09 30.74 5.71 5.75 Hollow (hep/pARG)2 capsules 0.349
Hollow (PSS/DAR)2 capsules 6.88 53.76 4.76 5.87 Hollow (PSS/DAR)2 capsules 1.118
Hollow (PSS/PDADMAC)2 capsules 4.33 48.87 7.31 7.49 Hollow (PSS/PDADMAC)2 capsules 0.755
Hep/pARG complexes 15.72 32.98 6.00 6.51 Hep/pARG complexes 0.351
PSS/DAR complexes 8.67 62.89 4.68 3.38 PSS/DAR complexes 0.511
PSS/PDADMAC complexes 4.79 56.23 8.44 8.50 PSS/PDADMAC complexes 0.775
1974 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977

Table 4 components, 2 periods which involved incubating the slugs on 1 ml

Influence of exposure to different test substances on mucus production and release PBS during 1 h were introduced. Subsequently, the liquid was
of proteins and LDH.
collected and analyzed for its protein and enzyme (LDH and ALP)
Treatment Total mucus Protein release LDH release content. The mean protein and LDH release of the 5 slugs of each
productiona
test group are shown in Table 4.
(mg/ml/g) (U/L/g) Although statistical analysis revealed no significant differences
DDWM 0.73  0.32c 38.00  52.83 ee in the mean protein release after exposure to the different test
DDWM/SLS (4/1) 4.73  1.85d 30.4  33.22 0.16  0.50 (1)x substances (Scheffé homogeneous subset, p-value >0.05), the
Heparinb 1.82  0.42d 27.4  22.23 ee
results of LDH release lead one to suspect that it is likely that
PSS∼PSS/DAR capsules, b 2.29  0.50d 24.7  17.29 ee
PSS∼PSS/PDADMAC capsules, b 2.43  0.57d 32.5  25.97 ee
DDWM/SLS (4/1), poly-L-arginine and DAR have the possibility to
Poly-L-arginineb 13.28  4.44d 45.1  25.03 0.58  1.20 (2)x cause tissue damage since these 3 components evoked LDH release.
PDADMACb 4.90  1.33d 37.7  24.20 ee ALP release was absent in the first and second sample for all test
DARb 8.33  2.86d 122.10  54.80 1.61  1.80 (4)x substances, indicating the absence of severe tissue damage in each
(PSS/PDADMAC)2 capsules 0.84  0.42c 38.6  37.95 ee
test group. Poly-L-arginine and DAR evoked LDH release however
(PSS/DAR)2 capsules 0.56  0.07c 27.00  18.22 ee
(hep/pARG)2 capsules 1.01  0.46c 33.4  34.49 ee this reaction was absent when complexing these positively charged
PSS/PDADMAC complexes 0.96  0.39c 22.4  15.60 ee polymers with negatively charged ones since no LDH release was
PSS/DAR complexes 1.13  0.20c 32.30  25.13 ee observed in case of complexes and hollow capsules composed of
hep/pARG complexes 0.70  0.41c 17.2  10.66 ee poly-L-arginine or DAR.
(x)x Amount of slugs which tested positive for LDH release.
Note: Data are presented as mean  standard deviation (n ¼ 5). 3.2.3. Slug survival
a
In % (w/w) of the initial body weight at the start of the contact period.
b After exposing the slugs to the different test substances, they
Polymer was supplemented with DDWM to obtain a total mass of 10 mg
according to Table 1. were kept in Petri dishes and supplied with protein rich food.
c,d
Mean values with the same superscript do not differ significantly from each Subsequently the mortality of the slugs in each test group was
other (Scheffé post-hoc test). evaluated 1, 2, 5 and 7 days post contact (Fig. 8). No mortality was
e
Value below detection limit.
observed 1 and 2 days post treatment. In the DAR group, some
remnants of DAR were still sticking to the body wall of the slugs,
which was not observed in any of the other groups. Five days post
Statistical analysis of the results of mucus production evoked by contact, mortality was observed in the (PSS/DAR)2 capsules,
the different treatments showed that the tested substances can be PSS∼PSS/PDADMAC and DAR groups. The slugs from the DAR group that
divided in 2 groups. All single polyelectrolyte components (heparin, were still alive at day 5, did not have DAR on their body wall
PSS, poly-L-arginine, PDADMAC and DAR) evoked a reaction anymore. However, their body wall was severely damaged since
comparable to the reaction of slugs placed on DDWM/SLS (4/1) grey areas of damaged cells were visible on their foot surface. Tissue
(positive control) (Scheffé homogeneous subset, p-value >0.05). By damage of the body wall of slugs was not observed for other test
contrast, polyelectrolyte multilayer capsules and polyelectrolyte substances. Seven days post contact, slug mortality was observed in
complexes made in solution gave rise to a reaction comparable to the DDWM/SLS (4/1), DAR, PSS∼PSS/PDADMAC and (PSS/DAR)2
the reaction of slugs placed on DDWM (negative control) (Scheffé capsules groups. In the DAR group only 1 out of 5 slugs was still
homogeneous subset, p-value >0.05). The slugs produced alive 7 days post contact, but the slug was severely harmed. The tail
a minimal amount of colorless mucus. of the slug was solid and deformed compared to a normal slug. This
When placing slugs on the single polycationic components (i.e. deformation was only visible 7 days after exposure. Moreover, the
pARG, PDADMAC and DAR), the slugs produced a large amount of foot surface of the slug showed grey areas indicating a severely
colored mucus. In case of DAR, the mucus produced by the slugs damaged slug body wall. In the other groups, tissue damage of the
had a brown color due to a mixture of a yellow color of the mucus body wall of the slugs could not be observed.
and green color of the polymer. The single polyanionic components Mortality of slugs within a period of 7 days post contact can be
(heparin and PSS) induced only moderate amounts of colored explained by the progressive continuation of tissue damage after
mucus production. These results show that both polyanions and exposure to the test substance. Although (PSS/DAR)2 capsules did
polycations evoked increased colored mucus secretion. However, not induce increased mucus secretion or release of proteins and
the polycations showed a tendency to be more irritating than the enzymes, high mortality was observed within a period of 1 week
polyanions. These results are in agreement with earlier published after exposure to the appropriate capsules. Therefore constructs
data demonstrating a more mucosal irritation after contact with containing DAR can be excluded from any further biomedical use.
positively charged molecules compared to negatively charged ones Contrary (PSS/PDADMAC)2 capsules and (hep/pARG)2 capsules can
[29]. Whereas single polyelectrolyte components induce increased be classified as being non-irritating towards mucosal membranes
and colored mucus secretion, these responses are dramatically since they did not evoke mucus production, protein and enzyme
reduced upon complexation with an oppositely charged poly- release, and mortality.
electrolyte, regardless if the polyelectrolytes were randomly com-
plexed in water or assembled in a controlled fashion in multilayer 3.2.4. Immunohistochemistry
capsules. For the purpose of drug delivery (hep/pARG)2 capsules hold
most potential as pARG-containing capsules have previously shown
3.2.2. Protein and enzyme release after exposure to test substances to be degradable upon internalization by phagocyting cells, both in
Serious damage of the single-layered epithelium and the vitro as well as in vivo [14,30e32]. Therefore we investigated in this
underlying connective tissue of the slug foot is characterized by section more thoroughly whether (hep/pARG)2 capsules did induce
increased release of proteins and enzymes such as lactate dehy- any tissue damage. For this purpose, slugs were exposed during 1 h
drogenase (LDH) and alkaline phosphatase (ALP). In case of tissue to 10 mg lyophilized (hep/pARG)2 capsules followed by immuno-
damage LDH will be first released since it is a cytosolic enzyme, histochemical evaluation of the foot of the slugs by making cry-
while ALP is a membrane-bound enzyme and will be released in osections followed by hematoxylin and eosin (H&E) staining
case of severe tissue damage. After exposing slugs to the test (Fig. 9).
 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977 1975

Fig. 8. Survival curves of slugs exposed to (A) single polyelectrolytes, (B) hollow polyelectrolyte multilayer capsules and (C) polyelectrolyte complexes made in solution.

The foot of a slug consists of outer single-layered epithelium
overlying connective tissue. Exposure of a slug to an irritating
component may lead to severely damaged epithelium cells which
can be observed through immunohistochemistry. Fig. 9A shows
an H&E stained cryosection of the foot of a control slug which
was not exposed to any test substance. The outer single
epithelium layer is continuous indicating absence of tissue
damage. The foot of slugs exposed to (hep/pARG)2 capsules
(Fig. 9B) showed also an intact outer epithelium layer compa-
rable to the control. These results further confirm on a micro-
scopic scale the relatively inert character of (hep/pARG)2
capsules regarding mucosal irritation.
1976 L.J. De Cock et al. / Biomaterials 32 (2011) 1967e1977
Fig. 9. Optical microscopy images of hematoxylin and eosin stained cryosections showing the foot of (A) a control, non-exposed, slug and (B) a slug exposed to (hep/pARG)2
capsules.
4. Conclusions
In this paper the mucosal irritation potency of several classes e
biopolymers, synthetic polyelectrolytes and reactive polyelectrolytes
e of oppositely charged polyelectrolytes, their complexes as well as
hollow multilayer capsules were evaluated via a A. lusitanicus slug-
based mucosal irritation assay. We found that notwithstanding the
fact that single (bio)polyelectrolyte components do show a tendency
to induce tissue irritation, this irritation is abolished upon
complexation with an oppositely charged polyelectrolyte, turning
the supramolecular structures into fairly inert species. In an era
where there is a lot of controversy concerning safety and toxicity
issues of nano- and micro-engineered materials, we demonstrate an
efficient method to assess tissue irritation using an in vivo platform
which does not involve vertebrates such as mice, rabbits or non-
human primates. Taken together, these findings put polyelectrolyte
capsules further en route towards drug delivery applications.
Acknowledgments
LJDC wishes to express her gratitude to the Institute for the
Promotion of Innovation by Science and Technology in Flanders (IWT-
Flanders) for their financial support. SDK acknowledges Ghent
University for a postdoctoral scholarship (BOF-GOA). BGDG
acknowledges the FWO-Flanders for a postdoctoral scholarship. AGS
acknowledges Prof. Helmut Möhwald for his continuous support.
Appendix
Figures with essential color discrimination. Figs. 1, 3, 8, and 9 in
this article are difficult to interpret in black and white. The full color
images can be found in the online version, at doi:10.1016/j.
biomaterials.2010.11.012.
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