Mucosal irritation potential of polyelectrolyte multilayer capsules

Biomaterials 32 (2011) 1967e1977

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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 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 b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 October 2010 Accepted 6 November 2010 Available online 3 December 2010

Polyelectrolyte multilayer capsules have recently gained interest as carriers for drug delivery. When envisioning mucosal administration, one is focused with potential concerns such as tissue irritation and tissue damage, induced by the carrier itself. In this paper we demonstrate the use of a slug-based (Arion 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 tissue irritation without using large numbers of vertebrates such as mice, rabbits or non-human primates. We found that although single polyelectrolyte components do induce tissue irritation, this response is dramatically reduced upon complexation with an oppositely charged polyelectrolyte, rendering fairly inert polyelectrolyte complexes. These findings put polyelectrolyte multilayer capsules further en route towards drug delivery applications. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Mucosa Microcapsule Drug delivery Heparin Biocompatibility

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) microparticles 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 semipermeable, 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: [email protected] (B.G. De Geest). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.11.012

high shear forces and (2) the ability to tailor the capsules’ physicochemical 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.

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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 PDADMAC (PSS/DAR)2 capsules PSS DAR (hep/pARG)2 capsules hep pARG

4.8 mg 5.0 mg 3.8 mg 6.1 mg 5.7 mg 4.3 mg

enzymes such as lactate dehydrogenase (LDH) and alkaline phosphatase (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) polystyrene-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 polyelectrolytes 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.

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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 polyelectrolyte 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 microparticles 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

2. Materials and methods

The composition of the respective polyelectrolyte complexes and capsules was determined by elemental analysis (Vario EL, Elementar Analysensysteme).

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.

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

Fig. 3. Schematic overview of the slug mucosal irritation test.

0.3 0.2

B

0

0.1 0.0 190

200

210

2 4 6 8 Number of bilayers

220 230 240 Wavelength [nm]

Absorbance [a.u.]

C

250

Absorbance [a.u.]

Absorbance [a.u.]

0.4

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

10

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2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 190

0.5 0.4 0.3 0.2 0.1 0.0 0

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0.5 0.4 0.3 0.2 0.1 0.0 0

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10

0.2 0.0 200

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350

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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 polyelectrolyte 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 containing 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 NanoOrange 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

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hollow

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0 -5 -10 -15 -20 -25 -30 -35 -40 2 3 Adsorption step

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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 multilayer build-up could be concluded. 3.1.2. Formation of polyelectrolyte films on sacrificial CaCO3 templates After having demonstrated the possibility to form polyelectrolyte multilayer films composed of hep/pARG, PSS/PDADMAC 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, especially 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 multilayer build-up was started with the polyanion. Due to their high porosity, CaCO3 microparticles strongly adsorb polymers, regardless their charge and previous experiments on CaCO3 microparticles (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 reorganization of the polyelectrolyte within the capsule shell upon dissolution 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 buildup in ambient light which is sufficient to induce coupling of both polymers [27].

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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 formation 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/PDADMAC 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 analysis 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

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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-Larginine 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 Table 2 Elemental analysis of hollow (hep/pARG)2, (PSS/DAR)2 and (PSS/PDADMAC)2 capsules and the corresponding complexes.

Hollow (hep/pARG)2 capsules Hollow (PSS/DAR)2 capsules Hollow (PSS/PDADMAC)2 capsules Hep/pARG complexes PSS/DAR complexes PSS/PDADMAC complexes

N(%)

C(%)

H(%)

S(%)

14.09 6.88 4.33 15.72 8.67 4.79

30.74 53.76 48.87 32.98 62.89 56.23

5.71 4.76 7.31 6.00 4.68 8.44

5.75 5.87 7.49 6.51 3.38 8.50

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 equipment, 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 application 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 3 Molar ratio of polyanion to polycation in hollow capsules and complexes composed of hep/pARG, PSS/DAR and PSS/PDADMAC. Molar ratio Hollow (hep/pARG)2 capsules Hollow (PSS/DAR)2 capsules Hollow (PSS/PDADMAC)2 capsules Hep/pARG complexes PSS/DAR complexes PSS/PDADMAC complexes

0.349 1.118 0.755 0.351 0.511 0.775

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Table 4 Influence of exposure to different test substances on mucus production and release of proteins and LDH. Treatment

Total mucus productiona

DDWM DDWM/SLS (4/1) Heparinb PSS∼PSS/DAR capsules, b PSS∼PSS/PDADMAC capsules, b Poly-L-arginineb PDADMACb DARb (PSS/PDADMAC)2 capsules (PSS/DAR)2 capsules (hep/pARG)2 capsules PSS/PDADMAC complexes PSS/DAR complexes hep/pARG complexes

0.73 4.73 1.82 2.29 2.43 13.28 4.90 8.33 0.84 0.56 1.01 0.96 1.13 0.70

             

0.32c 1.85d 0.42d 0.50d 0.57d 4.44d 1.33d 2.86d 0.42c 0.07c 0.46c 0.39c 0.20c 0.41c

Protein release

LDH release

(mg/ml/g)

(U/L/g)

38.00 30.4 27.4 24.7 32.5 45.1 37.7 122.10 38.6 27.00 33.4 22.4 32.30 17.2

             

52.83 33.22 22.23 17.29 25.97 25.03 24.20 54.80 37.95 18.22 34.49 15.60 25.13 10.66

ee 0.16  0.50 (1)x ee ee ee 0.58  1.20 (2)x ee 1.61  1.80 (4)x ee ee ee ee ee ee

(x)x Amount of slugs which tested positive for LDH release. Note: Data are presented as mean  standard deviation (n ¼ 5). a In % (w/w) of the initial body weight at the start of the contact period. b Polymer was supplemented with DDWM to obtain a total mass of 10 mg according to Table 1. c,d Mean values with the same superscript do not differ significantly from each other (Scheffé post-hoc test). e Value below detection limit.

Statistical analysis of the results of mucus production evoked by the different treatments showed that the tested substances can be divided in 2 groups. All single polyelectrolyte components (heparin, PSS, poly-L-arginine, PDADMAC and DAR) evoked a reaction comparable to the reaction of slugs placed on DDWM/SLS (4/1) (positive control) (Scheffé homogeneous subset, p-value >0.05). By contrast, polyelectrolyte multilayer capsules and polyelectrolyte complexes made in solution gave rise to a reaction comparable to the reaction of slugs placed on DDWM (negative control) (Scheffé homogeneous subset, p-value >0.05). The slugs produced a minimal amount of colorless mucus. When placing slugs on the single polycationic components (i.e. pARG, PDADMAC and DAR), the slugs produced a large amount of colored mucus. In case of DAR, the mucus produced by the slugs had a brown color due to a mixture of a yellow color of the mucus and green color of the polymer. The single polyanionic components (heparin and PSS) induced only moderate amounts of colored mucus production. These results show that both polyanions and polycations evoked increased colored mucus secretion. However, the polycations showed a tendency to be more irritating than the polyanions. These results are in agreement with earlier published data demonstrating a more mucosal irritation after contact with positively charged molecules compared to negatively charged ones [29]. Whereas single polyelectrolyte components induce increased and colored mucus secretion, these responses are dramatically reduced upon complexation with an oppositely charged polyelectrolyte, regardless if the polyelectrolytes were randomly complexed in water or assembled in a controlled fashion in multilayer capsules. 3.2.2. Protein and enzyme release after exposure to test substances Serious damage of the single-layered epithelium and the underlying connective tissue of the slug foot is characterized by increased release of proteins and enzymes such as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP). In case of tissue damage LDH will be first released since it is a cytosolic enzyme, while ALP is a membrane-bound enzyme and will be released in case of severe tissue damage. After exposing slugs to the test

components, 2 periods which involved incubating the slugs on 1 ml PBS during 1 h were introduced. Subsequently, the liquid was collected and analyzed for its protein and enzyme (LDH and ALP) content. The mean protein and LDH release of the 5 slugs of each test group are shown in Table 4. Although statistical analysis revealed no significant differences in the mean protein release after exposure to the different test substances (Scheffé homogeneous subset, p-value >0.05), the results of LDH release lead one to suspect that it is likely that DDWM/SLS (4/1), poly-L-arginine and DAR have the possibility to cause tissue damage since these 3 components evoked LDH release. ALP release was absent in the first and second sample for all test substances, indicating the absence of severe tissue damage in each test group. Poly-L-arginine and DAR evoked LDH release however this reaction was absent when complexing these positively charged polymers with negatively charged ones since no LDH release was observed in case of complexes and hollow capsules composed of poly-L-arginine or DAR. 3.2.3. Slug survival After exposing the slugs to the different test substances, they were kept in Petri dishes and supplied with protein rich food. Subsequently the mortality of the slugs in each test group was evaluated 1, 2, 5 and 7 days post contact (Fig. 8). No mortality was 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 contact, mortality was observed in the (PSS/DAR)2 capsules, PSS∼PSS/PDADMAC and DAR groups. The slugs from the DAR group that were still alive at day 5, did not have DAR on their body wall anymore. However, their body wall was severely damaged since grey areas of damaged cells were visible on their foot surface. Tissue damage of the body wall of slugs was not observed for other test substances. Seven days post contact, slug mortality was observed in the DDWM/SLS (4/1), DAR, PSS∼PSS/PDADMAC and (PSS/DAR)2 capsules groups. In the DAR group only 1 out of 5 slugs was still alive 7 days post contact, but the slug was severely harmed. The tail of the slug was solid and deformed compared to a normal slug. This deformation was only visible 7 days after exposure. Moreover, the foot surface of the slug showed grey areas indicating a severely damaged slug body wall. In the other groups, tissue damage of the body wall of the slugs could not be observed. Mortality of slugs within a period of 7 days post contact can be explained by the progressive continuation of tissue damage after exposure to the test substance. Although (PSS/DAR)2 capsules did not induce increased mucus secretion or release of proteins and enzymes, high mortality was observed within a period of 1 week after exposure to the appropriate capsules. Therefore constructs containing DAR can be excluded from any further biomedical use. Contrary (PSS/PDADMAC)2 capsules and (hep/pARG)2 capsules can be classified as being non-irritating towards mucosal membranes since they did not evoke mucus production, protein and enzyme release, and mortality. 3.2.4. Immunohistochemistry For the purpose of drug delivery (hep/pARG)2 capsules hold most potential as pARG-containing capsules have previously shown to be degradable upon internalization by phagocyting cells, both in vitro as well as in vivo [14,30e32]. Therefore we investigated in this section more thoroughly whether (hep/pARG)2 capsules did induce any tissue damage. For this purpose, slugs were exposed during 1 h to 10 mg lyophilized (hep/pARG)2 capsules followed by immunohistochemical evaluation of the foot of the slugs by making cryosections followed by hematoxylin and eosin (H&E) staining (Fig. 9).

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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 comparable to the control. These results further confirm on a microscopic scale the relatively inert character of (hep/pARG)2 capsules regarding mucosal irritation.

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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 slugbased 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 nonhuman primates. Taken together, these findings put polyelectrolyte capsules further en route towards drug delivery applications.

[4] [5]

[6] [7]

[8]

[9] [10] [11] [12] [13]

Acknowledgments LJDC wishes to express her gratitude to the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWTFlanders) 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.

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Appendix

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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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