Cytotoxicity of monodispersed chitosan nanoparticles against the Caco-2 cells

Toxicology and Applied Pharmacology 262 (2012) 273–282

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Cytotoxicity of monodispersed chitosan nanoparticles against the Caco-2 cells Jing Wen Loh a, Martin Saunders b, Lee-Yong Lim a, c,⁎ a b c

Laboratory for Drug Delivery, Pharmacy, Characterisation and Analysis, University of Western Australia, Australia Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Australia School of Biomedical, Biomolecular and Chemical Sciences, 35 Stirling Hwy, Crawley 6009, Australia

a r t i c l e

i n f o

Article history: Received 11 February 2012 Revised 25 April 2012 Accepted 30 April 2012 Available online 18 May 2012 Keywords: Chitosan nanoparticles Intestinal cells Necrosis Toxicity

a b s t r a c t Published toxicology data on chitosan nanoparticles (NP) often lack direct correlation to the in situ size and surface characteristics of the nanoparticles, and the repeated NP assaults as experienced in chronic use. The aim of this paper was to breach these gaps. Chitosan nanoparticles synthesized by spinning disc processing were characterised for size and zeta potential in HBSS and EMEM at pHs 6.0 and 7.4. Cytotoxicity against the Caco-2 cells was evaluated by measuring the changes in intracellular mitochondrial dehydrogenase activity, TEER and sodium fluorescein transport data and cell morphology. Cellular uptake of NP was observed under the confocal microscope. Contrary to established norms, the collective data suggest that the in vitro cytotoxicity of NP against the Caco-2 cells was less influenced by positive surface charges than by the particle size. Particle size was in turn determined by the pH of the medium in which the NP was dispersed, with the mean size ranging from 25 to 333 nm. At exposure concentration of 0.1%, NP of 25 ± 7 nm (zeta potential 5.3 ± 2.8 mV) was internalised by the Caco-2 cells, and the particles were observed to inflict extensive damage to the intracellular organelles. Concurrently, the transport of materials along the paracellular pathway was significantly facilitated. The Caco-2 cells were, however, capable of recovering from such assaults 5 days following NP removal, although a repeat NP exposure was observed to produce similar effects to the 1st exposure, with the cells exhibiting comparable resiliency to the 2nd assault. © 2012 Elsevier Inc. All rights reserved.

Introduction Chitosan nanoparticles are one of the most extensively studied materials in the biomedical industry and are highly regarded as a material of choice in drug delivery applications (Ajun et al., 2009; Chen et al., 2008; Cheng et al., 2009; Fernández-Urrusuno et al., 2004). The objectives are often to reduce drug side effects, control the rate of drug delivery, and ensuring only the targeted area is treated (Elzatahry and Mohy-Elidin, 2008; Mitra et al., 2001). The use of chitosan nanoparticles in drug delivery also has the added benefit of increasing drug permeation through the absorptive epithelia (Bejugam et al., 2008; Smith et al., 2004). While the chitosan parent polymer is generally regarded to be safe and biocompatible, it is not unusual to find embedded in these studies evidence of the cytotoxicity caused by chitosan nanoparticles (Liang et al., 2011; Loretz and Bernkop-Schnurch, 2007; Qi et al., 2005). Using lung and intestinal epithelial cell models, previous studies (Huang et al., 2004; Ma and Lim, 2003) observed that the chitosan nanoparticles (122 ± 5 nm) exhibited different mechanisms of cellular uptake and distribution in comparison to chitosan, which were presented as dissolved molecules. Recently, Loh et al. (2010a) ⁎ Corresponding author at: School of Biomedical, Biomolecular and Chemical Sciences, 35 Stirling Hwy, Crawley 6009, Australia. Fax: + 61 8 6488 7532. E-mail address: [email protected] (L.-Y. Lim). 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2012.04.037

developed a capacity for synthesising smaller chitosan nanoparticles with narrow size distribution (18 ± 1 nm) by employing the spinning disc processing technology (SDP). Subsequent testing of these nanoparticles indicated that the cell membrane integrity of human hepatocytes has been compromised, causing necrotic and/or autophagic cell death (Loh et al., 2010b). The chitosan nanoparticles have also caused a dose-dependent increase in CYP3A4 enzyme activity in the human hepatocytes. These results led us to question whether the smaller chitosan nanoparticles would have a similar toxic effect on intestinal cells. The evaluation of drug toxicity against the intestinal mucosal is important to the pharmaceutical industry as the oral pathway is the preferred route for the administration of drugs. The Caco-2 cell line is often used as the surrogate of the human intestine since it spontaneously differentiates after 3–4 weeks into highly polarised enterocyte-like phenotype (Rodriguez-Juan et al., 2001) with functional tight junctions (Leonard et al., 2000) when cultured on porous membranes under normal culture conditions. The differentiated cells developed well organised microvilli on the apical membrane, and expressed many of the enzymes and transporters found in absorptive enterocytes (Miret et al., 2004). For these reasons, the Caco-2 cell line was selected to investigate the cytotoxicity of chitosan nanoparticles produced by the SDP. Apart from nanoparticle cytotoxicity, there are also a number of published studies (e.g., Hafner et al., 2009; Loretz and Bernkop-Schnurch, 2007) on the interaction of chitosan nanoparticles with Caco-2 cells. In

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most cases, the experiments were conducted in acidic aqueous media (pHb 6.5) to facilitate the ionisation of the amino groups in the chitosan polymer. The resultant positive charges enabled the chitosan nanoparticles to associate with the negatively charged sialic acid residues in mucus, thereby imparting a mucoadhesive property (de Campos et al., 2004). However, cell membrane damage could also have resulted from the electrostatic interactions between the chitosan nanoparticles and cell membrane, as demonstrated in a study by Loretz and BernkopSchnurch (2007), which involved chitosan nanoparticles of 197±54 nm with a zeta potential of +31.0±1.5 mV. Additionally, cellular uptake of chitosan nanoparticles (433 ± 28 nm, + 27.2 ± 0.8 mV, in HBSS at pH 5.5) by clathrin-mediated endocytosis has been demonstrated, with twice the amount of nanoparticles found in the Caco-2 cells after 2 h exposure at 0.1% loading concentration when compared to chitosan molecules (Ma and Lim, 2003). Moreover, chitosan molecules (Dodane et al., 1999) and chitosan nanoparticles (Loretz and Bernkop-Schnurch, 2007) have been shown to induce a temporal increase in tight junction permeability in a concentration-dependent manner in Caco-2 cell monolayers. These studies have also demonstrated the reversibility of the tight junctions in Caco-2 cells exposed to a single dosing of chitosan nanoparticles. The mechanism appeared to have involved a partial modulation of the cytoskeleton through induced changes in the cellular actin filaments (Dodane et al., 1999). Despite the plethora of studies (e.g., Ge et al., 2009; Kompella et al., 2003; Loretz and Bernkop-Schnurch, 2006; Qi et al., 2005), most have not provided any clear evidence of the correlation between cells and nanoparticle characteristics. The majority of these studies employed polydispersed chitosan nanoparticles with mean diameter above 65 nm and size distribution range between 37 nm and 100 nm, making it difficult to attribute the cellular interactions to any specific particle size. Moreover, the particle characteristics were not measured in the medium used for the cell culture experiments. Therefore, the objectives of this paper are (1) to characterise chitosan nanoparticles in biorelevant media to provide closer correlation of their toxicity profile to their in situ characteristics at the biological interfaces; (2) to evaluate the biological effects of chitosan nanoparticles against the Caco-2 cells upon repeat exposure to provide a more realistic evaluation of the nanoparticles in clinical use. Materials and methodology Materials. Chitosan (medium molecular weight), phosphate buffered saline (PBS; 0.138 M NaCl, 0.0027 M KCl; 0.0015 M KH2PO4; 0.0081 M HNa2PO4; pH 7.4), N-acetyl-D-glucosamine, Hank's balanced salt solution (HBSS), sodium fluorescein (NaF), thiazolyl blue tetrazolium bromide (MTT), dextran sulphate sodium salt, sodium dodecyl sulphate (SDS), isopropanol, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), glutaraldehyde, tannic acid, Earle's Minimum Essential Medium (EMEM), non-essential amino acids, penicillin, streptomycin and glutamine were purchased from Sigma-Aldrich (New South Wales, Australia). Foetal bovine serum was purchased from Gibco (Victoria, Australia). Spurrs® resin and osmium tetroxide (1% in PBS) were provided by the Centre for Microscopy, Characterisation and Analysis (UWA, Western Australia, Australia). Transwell® permeable supports were purchased from Corning® Inc. (24-well, 6.5 mm diameter, 0.4 μm pore size, polycarbonate membrane, New York, USA). Peel-A-Way® embedding moulds were purchased from Proscitech (22 × 22 mm, high density polyethylene, Queensland, Australia). All other cell culture plates and flasks were purchased from Greiner Bio One (Neuburg, Germany). Test samples. Chitosan nanoparticles were produced by ionotropic gelation using the SDP. Chitosan molecules (Cs) and nanoparticles (NP) were prepared and isolated according to the methods outlined in Loh et al. (2010a). Fluorescein isothiocyanate-labelled Cs and NP (FITC-Cs and FITC-NP, respectively) were also prepared and

characterised according to the previously published methods (Loh et al., 2010b). Test samples were dispersed in HBSS or supplemented EMEM transport medium at a concentration range of 0.01%–0.1%, then adjusted to pH 7.4 or pH 6.0 with 3 M HCl. The samples were characterised by shape, size and zeta potential using dynamic light scattering (DLS) and laser Doppler anemometry techniques, respectively (Zetasizer Nano ZS, Malvern Instruments, Version 4, Worcs., UK). Cell culture. Caco-2 cells (passage 40) purchased from the European Collection of Cell Cultures (ECACC, Salisbury, Great Britain) were grown in 75 cm 2 culture flasks at 37 °C in 5% CO2 and 90% relative humidity (Thermo forma series 2, Ohio, USA) in 10 mL of EMEM supplemented with 1% non-essential amino acids, 10,000 U/mL penicillin, 10 mg/mL streptomycin, 200 mM glutamine and 10% foetal bovine serum. Spent culture medium was exchanged every 2–3 days with 10 mL of fresh medium and sub-cultured at >80% confluency for experiments. Cells between passages 46 and 66 were used for subsequent experiments. Mitochondrial dehydrogenase activity (MDA). The analysis of MDA provides an overview of the cytotoxicity inflicted by the test samples. In this study, MDA analysis was also used to determine the length of exposure time (h) for subsequent experiments. Caco-2 cells were plated in 96-well plates at 10,000 cells per well and cultured over 48 h with 100 μL of supplemented EMEM. The spent medium was aspirated and the cells were incubated with samples at pH 7.4 or pH 6.0 for 4–72 h at 37 °C in 5% CO2 and 90% relative humidity. Experiments up to 24 h incubation used HBSS as the vehicle. Experiments involving 48 or 72 h exposure used culture medium as the vehicle, as the cells could not survive for long periods in HBSS. For the 72 h exposure experiments, the samples were removed at 48 h, the cells were washed once with pre-warmed culture medium (100 μL) and incubated with corresponding fresh Cs or NP samples for a further 24 h. After the specified exposure times, the cells were washed once with 100 μL of pre-warmed PBS before they were incubated for 2 h with 100 μL of MTT solution (1 mg/mL in PBS). Following the aspiration of the MTT solution, the cells were solubilised with 100 μL of 10% w/v SDS (0.01 M HCl in isopropanol as vehicle) overnight to extract the intracellular formazan crystals. The colour of the cell lysate was measured at 570 nm (BioTek EL808, Vermont, USA) and the results were expressed as a percentage activity relative to cells exposed to the corresponding blank vehicles. Dextran sulphate sodium salt (0.1% w/v) and SDS (0.1% w/v) in corresponding vehicles were used as positive and negative controls, respectively. Transport studies. Transport studies examine the capacity of chitosan to modulate the tight junctions in differentiated Caco-2 cells. The cells at a seeding density of 10,000 cells/well were grown on the apical chamber of 24-well Transwell® permeable supports in 0.1 mL of pre-warmed EMEM with 0.6 mL of EMEM in the basal compartment. Spent medium was exchanged every other day. After 25 days of culture, the cells were prepared for transport experiments by washing once with pre-warmed HBSS (pH 6.0), and equilibrated with prewarmed HBSS (0.1 mL apical, 0.6 mL basal) for 30 min in the CO2 incubator. The integrity of the cell monolayers was monitored through the measurement of transepithelial electrical resistance (TEER) using the MilliCell®-ERS Chopstick Electrodes (Millipore, Massachusetts, USA). Cells with a net TEER ≥200 Ω.cm 2 were used for subsequent transport experiments. Samples of Cs or NP were prepared in pH 6.0 HBSS medium containing 0.01% of sodium fluorescein (NaF, 37.6 M). The transport medium (HBSS containing 0.01% w/v NaF) served as the negative control. To start the transport experiments, the medium in the basal chamber was replaced with fresh pre-warmed HBSS while that in the apical chamber was replaced with 0.1 mL of pre-warmed samples.

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After predetermined periods (30, 60, 120 and 180 min) of incubation at 37 °C in 5% CO2/95% relative humidity, 200 μL of medium in the basal compartment was withdrawn for NaF analysis at 490 nm. The withdrawn aliquot was immediately replaced with an equal volume of fresh pre-warmed HBSS to maintain media volume in the basal chamber. At the final time point of sampling, the NaF content in 200 μL of medium from the apical compartment was also measured. Immediately after the last time point of sampling, the cells were washed once with HBSS before they were returned to the CO2 incubator with pre-warmed EMEM. During this recovery period, the spent medium was exchanged once. On the 5th day, the tight junction integrity of the cell monolayers was assessed in the transport medium. The next day, the cells were subjected to a second cycle of exposure to the Cs and NP samples followed by a recovery period using the same experimental conditions as described for the first cycle. Laser scanning confocal microscopy. Cs and NP uptakes into Caco-2 cell monolayers were evaluated using laser scanning confocal microscopy. Caco-2 cells (2 × 10 5) were seeded onto a glass cover (#1 thickness) placed in a 22-mm Petri dish with 1.5 mL of EMEM. At full confluency (48 h), the spent medium was replaced with 1.5 mL of FITC-Cs or FITC-NP dispersion (0.1% w/v in pH 6.0 HBSS). After 4 h of incubation, the cells were washed twice with PBS, fixed in paraformaldehyde (4% w/v in PBS) for 15 min, and then washed thrice with PBS. Viable cells were differentiated by nuclear staining with DAPI (2 mM in PBS) for 10 min, the excess stain removed by washing with PBS for 1 min before the cover slip was mounted onto a clean slide for viewing under the laser scanning confocal microscope (Leica TCS SP2, Illinois, USA). FITC-Cs and FITC-NP were detected at λex: 488 nm, λem: 536–624 nm while the DAPI fluorescence was detected at a lower λex: 385 nm and λem: 400–480 nm. Transmission electron microscopy (TEM). TEM is used to assess the morphology of Caco-2 cell monolayers following exposure to the test samples. Caco-2 cells (200,000 cells) were seeded on Peel-AWay® embedding moulds with 1.5 mL of culture medium and grown for 4 days before incubation with 1 mL of the test samples in transport medium. HBSS at pH 6.0 served as the transport medium as well as the negative control. After 4 h, the samples were removed and the cells were washed once with 1.5 mL of PBS before overnight incubation at ambient temperature with 1.5 mL of prefix solution (2.5% glutaraldehyde and 1% tannic acid in PBS). Then the cells were stained with 1% osmium in PBS and embedded in Spurrs® resin for viewing under the TEM (JOEL JEM 2100, Tokyo, Japan) at 120 kV with a spot size of 1 and alpha 3. Statistical analysis. Data are expressed as mean ± standard deviation obtained from ≥3 independent experiments conducted on different days with ≥1 repeat wells per experiment. Statistical analysis of data was performed using analysis of variance (ANOVA) with posthoc Tukey's test for paired comparison of the means (SPSS Version 11, Lead Technologies Inc., Chicago, USA). Results Test sample properties Cs and NP characteristics differed in the two media and at the two pHs used (Table 1). NP particle size as measured by the DLS was about 6 to 13 fold larger at pH 7.4 than at pH 6.0, indicating significant particle aggregation at neutral pH, particularly in the HBSS medium. At pH 7.4, there was a 2-fold increase in particle size when the medium was switched from EMEM to HBSS, whereas comparable NP particle sizes were observed at pH 6 in both media. These changes in particle size did not correlate well with the zeta potential values measured in the respective media. While relatively low zeta potential values were

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Table 1 Size and zeta potential (ZP) for Cs and lyophilised NP reconstituted at 0.1% w/v in various biorelevant media. *symbolises particle sizes exceeding the upper limit of the instrument. Data represent mean size ± standard deviation (n > 3). Media

Chitosan pH 7.4

Nanoparticles pH 6.0

pH 7.4

pH 6.0

Particle size (nm) HBSS 1817 ± 309 EMEM *

1809 ± 171 1558 ± 301

333 ± 43 149 ± 16

25 ± 7 26 ± 2

Zeta potential (mV) HBSS − 5.4 ± 0.9 EMEM − 8.5 ± 0.8

9.9 ± 1.9 8.8 ± 1.0

3.3 ± 0.4 − 1.4 ± 0.2

5.3 ± 2.8 − 6.1 ± 1.9

registered for the NP in both media, there were clear trends of positive values in HBSS and negative values in EMEM, with an increase in zeta potential magnitude upon acidification of the respective media to pH 6.0. Cs particle size was much higher and consistently above 1 μm at both pHs in the two media. Unlike the NP particles, the zeta potential of the Cs particles was less affected by the composition of the biorelevant medium than by their pH, exhibiting negative charges at pH 7.4 and positive charges at pH 6.0. Mitochondrial dehydrogenase activity MDA activity of the Caco-2 cells was evaluated after 4, 24, 48 and 72 h exposure at pH 6.0 and pH 7.4 (Fig. 1). As with many published in vitro cytotoxicity studies, to ensure cell survival, the experiments were performed with the HBSS medium at 4 and 24 h exposures, and with the cell culture medium, EMEM, at 48 and 72 h exposures. One striking feature of the cytotoxicity profiles shown in Fig. 1 is the relative innocuity of the Cs samples at the range of concentrations and time points tested. Except for cells exposed for 48 h to 0.1% Cs in EMEM (Fig. 1, 3B), which had cell viability slightly below 80%, the Caco-2 cells were observed to be generally resilient against Cs exposure. Unlike the Cs samples, the NP samples showed low cytotoxicity profiles only at pH 7.4. At this pH, for all concentration ranges and time points tested, the NP did not lower the viability of the Caco-2 cells to below 80%, except for 0.1% NP at 4 h exposure, where cell viability of about 60% was obtained (Fig. 1, 1A). On the other hand, when the experimental pH was lowered to 6.0, cell viability below 40% was observed for 0.1% NP at all exposure times evaluated, as well as for 0.05% NP at 4 and 24 h exposures. Transport studies Transport studies were performed using the pH 6.0 HBSS medium, in which the dispersed NP sample had mean size of 25 nm and positive zeta potential of 5.3 mV, while the Cs sample had mean size of 1809 nm and zeta potential of 9.9 mV. Tight junction permeability in the Caco-2 cell monolayers was monitored by TEER measurements (Fig. 2) and sodium fluorescein transport data (Fig. 3). TEER recorded was calculated as a percentage relative to baseline TEER (measured just prior to experiment initiation) and plotted against exposure time. Over a 3 h period of exposure to Cs, the TEER values of the Caco-2 cell monolayers remained above 84% of the baseline value, similar to those observed in cells exposed to HBSS (Fig. 2 IA). Exposure to a second sample of Cs, following 5 days of recovery in EMEM after the 1st Cs exposure, produced a similar TEER profile in the Caco-2 cells (Fig. 2 IIA), the TEER values remaining above 83% at all concentrations studied. The retention of tight junction integrity during both Cs exposures and the respective recovery periods was confirmed by the fluorescein transport data (Fig. 3A). The Papp values

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Fig. 1. Mitochondrial dehydrogenase activity of Caco-2 cells exposed to Cs and NP samples for (1A) 4 h at pH 7.4; (1B) 4 h at pH 6.0 (2A) 24 h at pH 7.4; (2B) 24 h at pH 6.0; (3A) 48 h at pH 7.4; (3B) 48 h at pH 6.0; (4A) 72 h at pH 7.4; and (4B) 72 h at pH 6.0. Panels 1–2 used HBSS as the transport medium and panels 3–4 used supplemented culture medium as the transport medium. Enzyme activity is calculated as a percentage relative to that of cells exposed to transport medium, and the values are expressed as mean ± standard deviation, n = 3. a denotes significant difference to dextran (negative control) and b denotes significant difference to chitosan at p b 0.05.

obtained were comparable to those for the negative control cells even at the highest applied concentration of 0.10% Cs (p > 0.05). On the other hand, the NP samples led to concentrationdependent decreases in TEER within 0.5 h of exposure, with TEER falling to 86%, 32% and 24% at exposure concentrations of 0.01%, 0.05% and 0.1%, respectively (Fig. 2 IB). The effect stabilised after 2 h with the TEER values recorded at 24%, 5% and 3%, for the respective concentrations at 3 h. Despite the sharp fall in TEER values, a corresponding increase in fluorescein transport was observed only at the highest NP concentration of 0.1% (Fig. 3B). It is not unusual that the trends in TEER and Papp values did not develop in parallel as they reflected different functional properties (Madara, 1998). Mean fluorescein Papp, measured after 3 h of transport in the presence of 0.1% of NP was significantly higher (7.5 ± 1.6 × 10 − 7 cm/s) than the value obtained for control cells (2.5 ± 1.2 × 10 − 7 cm/s; p = 0.023).

However, the NP-induced changes in TEER and fluorescein Papp were temporary, even for those exposed to 0.1% of NP. The cell monolayers, after 5 days of incubation with EMEM post-NP exposure, were found to exhibit TEER comparable to those measured in the negative control cells (Fig. 2 ID). This recovery in tight junction integrity was supported by fluorescein Papp values (Fig. 3B), which were no different from those obtained in control cells. The 2nd dose of NP caused the TEER to fall lower than that measured with the 1st NP dose (Fig. 2 IID). TEER fell to 55%, 17% and 9% after 0.5 h exposure to 0.01%, 0.05% and 0.1% of NP, respectively. It fell further to b6% of baseline TEER after 3 h at all 3 NP concentrations. Once more, the fluorescein transport data did not reflect the dramatic drop in TEER, as the fluorescein Papp values obtained for the 2nd dose of NP were similar to those recorded for the 1st NP dose (Fig. 3B). Similarly, only those cells exposed to 0.1% of NP

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Fig. 2. Changes in TEER measured in Caco-2 cell monolayers exposed to Cs and NP samples in pH 6.0 HBSS medium. TEER was monitored during exposure to the 1st doses (I) of Cs (A) and NP (B) over 3 h; the samples were then removed, and cells were allowed to recover in EMEM for the next 5 days. On the 5th day, TEER was measured for 3 h during a fluorescein transport experiment conducted in the cells previously exposed to Cs (C) and NP (D). On the 6th day, the 2nd doses of Cs and NP were administered (II) and TEER monitoring was repeated using the same protocol as the first dose. Results (mean ± SD, n > 4) are expressed as a percentage of baseline TEER, measured just prior to experiment commencement.

showed significantly higher Papp, at 6.4 ± 1.3 (×10 − 7 cm/s), than the negative control cells. Remarkably, the changes in cell monolayer integrity associated with the 2nd dose of NP were also reversible following 5 days of incubation

with the culture medium after NP removal. At all concentrations assessed, the TEER values were increased to ≥92% of baseline (Fig. 2 IID), similar to the first recovery profile, and the fluorescein Papp values were comparable to those of the negative control cells (Fig. 3B).

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Figs. 5G and H showed extensive fragmentation of intracellular structures within the Caco-2 cells, accompanied by the condensation of the cell nucleus following exposure to 0.1% of NP. In contrast, the same concentration of Cs did not result in significant changes to the cell morphology (Figs. 5C and D) compared to the control cells (Figs. 5A and B). Salient subcellular structures, such as the mitochondria, could be clearly delineated in the micrographs. Exposure to a much lower NP concentration of 0.005% did not result in the extensive damage evident in cells exposed to 0.1% NP. However, there was an apparent increase in the number of mitochondrial organelles, particularly in close proximity to the cell nucleus, in cells exposed to 0.005% of NP (Figs. 5E and F).

Discussion

Fig. 3. The apparent permeability coefficient (Papp) values obtained for sodium fluorescein transport. Dose 1 represents cells exposed to the 1st cycle of Cs (A) or NP (B) at pH 6.0. Recovery 1 represents data obtained from the same cells grown in culture medium for 5 days following 1st exposure. Dose 2 represents data obtained from the cells exposed to the 2nd cycle of Cs or NP after the recovery period. Recovery 2 represents data obtained from cells grown in culture medium for 5 days following 2nd exposure. Results are expressed as mean ± standard deviation (n ≥ 3); * indicates statistical difference compared to HBSS.

Laser scanning confocal micrographs Localisation of cell-associated Cs and NP was detected at λex: 488 nm, λem: 536–624 nm following 4 h exposure of the Caco-2 cell monolayers to 0.1% of FITC-Cs and 0.1% FITC-NP in pH 6.0 HBSS (Fig. 4). Analysis of the confocal micrographs suggested that the Caco-2 cell monolayers grown on the cover slips were approximately 10 μm in thickness. To differentiate between particles attached on the surface of the cell and those internalised into the cells, micrographs of the cell surface and at a depth of 5 μm from the cell surface were taken. Micrographs of the cell surface showed strong fluorescent signals for cells exposed to FITC-Cs (Fig. 4A) whereas only faint fluorescence was detected for cells exposed to FITC-NP (Fig. 4B). In contrast, when the analysis was focused at a depth of 5 μm from the cell monolayer surfaces, minimal fluorescence was detected for cells exposed to FITC-Cs (Fig. 4C) while intense fluorescent signals were detected for the FITC-NP-exposed cell samples (Fig. 4D). Staining of the cell nucleic acids with DAPI allowed for visualisation of the cell nucleus at a lower wavelength (λex: 385 nm and λem: 400–480 nm) than that for the FITC. An overlay of the images produced from the DAPI (blue) and FITC (green) signals (Fig. 4E) showed extensive colocalisation of the DAPI and FITC, confirming the presence of NP in the nucleus of the Caco-2 cells. Transmission electron micrographs TEM was used to detect changes in the cell ultrastructures after incubation with Cs at 0.1%, and NP at 0.005% and 0.1% in pH 6.0 HBSS. Two representative micrographs per cell sample are shown in Fig. 5.

The results of this study showed that the Caco-2 cells were more sensitive to the cytotoxicity of NP at pH 6.0 than at pH 7.4 for all the exposure times assessed by MDA (Fig. 1). The same phenomenon was observed for chitosan molecules and nanoparticles in a number of studies (Loretz and Bernkop-Schnurch, 2007; Mohy Eldin et al., 2008; Yang et al., 2009), and was often attributed to chitosan possessing higher positive surface charges due to more extensive protonation of its amino groups at pH below its pKa. This explanation is, however, disputable in the present study, as the zeta potentials measured in HBSS between pH 6.0 and pH 7.4 were not significantly different for the NP sample (Table 1). In addition, cytotoxicity was observed for the 0.1% NP sample dispersed in EMEM (48 and 72 h exposure) at pH 6.0, when the NP possessed negative zeta potentials. The collective data suggest that the size of NP is a more plausible explanation for the differences in cytotoxicity observed for NP at pH 6.0 (25 ± 7 nm) since they were significantly smaller than NP at pH 7.4 (333 ± 43 nm). By itself, lower pH was not expected to affect cell viability in the short term as Caco-2 cells had been demonstrated to withstand pH as low as 5.7 for up to 2 h (Loretz and Bernkop-Schnurch, 2007). However, the motility and proliferation of Caco-2 cells were reported to have decreased by as much as 31.4% after 48 h following a change in culture medium pH from 7.4 to 7.0 (Perdikis et al., 1998). A similar predisposition to optimal growth and function at pH 7.4 might exist for the Caco-2 cells in this study, as it was observed that cells exposed for >4 h to blank pH 6.0 media, be it HBSS or culture medium, were of a lighter colour compared to cells exposed to corresponding media at pH 7.4. The cells at pH 6.0 might therefore be more susceptible to the effects of NP, although, based on the MDA, they remained resilient to the presence of Cs, even after 72 h of co-incubation. The low cytotoxicity of Cs could not be attributed to a lack of positive charges on the molecules, as the Cs samples exhibited positive zeta potentials, which reached almost + 10 mV, in the HBSS and EMEM media at pH 6.0. More likely, the relative lack of cytotoxicity was associated with the micron size particles obtained when Cs was dispersed in either media at the two pHs used. Micron sized particles are known to be effectively excluded from cellular uptake into the cytoplasm, and this was confirmed by electron micrographs in this study. In agreement with previous studies by our laboratory employing larger chitosan nanoparticles (110 nm – 390 nm) (Huang et al., 2004; Ma and Lim, 2003), the poorer cellular internalisation of Cs might account for the different cellular responses of the Caco-2 cells towards the Cs and NP samples. Unlike the NP, which was shown by confocal microscopy to be extensively internalised by the cells, Cs was poorly taken up into the cytoplasm despite strong adherence to the cell surface. The poor cellular internalisation of Cs was likely due to its large size (≫ 1 μm) in the two media. It is interesting to note that, while the current study showed that Cs at up to 0.1% did not affect the viability of the Caco-2 cells as measured by MDA, it

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Fig. 4. Confocal micrographs of Caco-2 cell monolayers after 4 h incubation with 0.1% of FITC-Cs (A and C) or 0.1% of FITC-NP (B and D). Cells were imaged at the cell surface (A, B) and 5 μm below the cell surface (C, D). Image E depicts cells incubated with 0.1% of FITC-NP with the cell nucleus co-stained blue with DAPI. All scale bars = 20 μm.

was shown to promote cytoproliferation of the A549 cells in an earlier study by our laboratory (Huang et al., 2004). Most cytotoxicity studies of chitosan were carried out over 4 h (Braydich-Stolle et al., 2005; Haas et al., 2005; Huang et al., 2004), which would not allow the evaluation of cellular responses to prolonged chitosan exposures, a hallmark of chronic therapy where drugs are administered in repeat doses. In this study, the MDA data suggests that a prolongation of NP exposure to 72 h did not produce additional toxicity to the Caco-2 cells at pH 6.0, particularly when the NP loading concentration was not more than 0.05%. In fact there appears to be a recovery of cell viability for the 0.025% NP sample in HBSS when the exposure time was prolonged from 4 to 24 h (Figs. 1, 1B vs 2B). These results are not in agreement with the findings of Qi et al. (2005), who observed chitosan nanoparticles to be more cytotoxic and exhibited a 3-fold reduction in IC50 value from 16.2 to 5.3 μg/mL when the incubation time was extended from 24 to 48 h in the MGC803 cells. Underlying reasons could be the differences in cell type, as well as the nanoparticle characteristics, for the chitosan nanoparticles prepared by Qi et al. (2005) had larger

diameter of 65 nm. Another reason might be that the Caco-2 cells responded to the NP assault by increasing intracellular mitochondria expression, as was observed in the TEM micrographs of cells exposed to 0.005% NP (Figs. 5E and F). However, as the concentration of 0.005% was substantially lower than the concentrations of NP used in the MDA assessment, further confirmatory experiments are required, since there is also no published report correlating internalised chitosan nanoparticles to the promotion of mitochondrial expression in cell cultures. Published paracellular transport studies involving chitosan nanoparticles have focused on single exposure to the nanoparticles (Bejugam et al., 2008; Ranaldi et al., 2002; Smith et al., 2004); again, this would not provide a realistic representation of the NP in use. Our data showed that, following co-incubation with the first dose of NP at 0.1%, the paracellular transport of sodium fluorescein was significantly facilitated, but the effect was reversible within days of removing the NP. This observation was consistent with published data, one of which showed TEER values to be restored 14 h after the removal of chitosan nanoparticles (Dodane et al., 1999;

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Fig. 5. Transmission electron micrographs showing the morphology of Caco-2 cells after 4 h incubation with HBSS (A, B), 0.1% of Cs (C, D), 0.005% of NP (E, F) and 0.1% of NP (G, H). Arrowheads depict cell mitochondria.

Loretz and Bernkop-Schnurch, 2007). We have, however, evaluated the effects of a second NP exposure and the subsequent cell recovery. The stronger cellular response to the 2nd dose of NP, based on the TEER data, implies that the cells had only partially recovered from the assault of the 1st NP dose. This is unsurprising given that the degree of reversibility of cellular response is inversely proportional to the magnitude of change induced (Ferruzza et al., 1999; Hurni et al., 1993). Nevertheless, even the changes induced by the 2nd NP dose

were also reversible, which is rather remarkable considering that the TEER of the Caco-2 cell monolayers was brought below 10% of baseline levels by the second NP assault. The integrity of the tight junctions, as represented by the TEER profiles, is modulated by cytoskeletal proteins such as actin and tubulin found in the apical membranes of differentiated Caco-2 cells (De Angelis et al., 1998; Dodane et al., 1999; Sambruy et al., 2001). Published studies to date have indicated that dissolved chitosan

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consistently disrupts intercellular tight junctions (Artursson et al., 1994; Schipper et al., 1999; Smith et al., 2004), possibly by redistributing such proteins to the cytoskeleton (Smith et al., 2004). Data from this study did not mirror this effect, as the paracellular fluorescein transport profiles of Cs-exposed cells were similar to the control cells (Fig. 3A). The anomaly might have arise from the lower degree of deacetylation of Cs used in this study (79 ± 2%) compared to the chitosans reported in the literature (>85%) (Artursson et al., 1994; Schipper et al., 1999; Smith et al., 2004). It was reported that the paracellular permeability can be reduced from 1.2 × 10 − 6 cm/s to as much as 5 × 10 − 7 cm/s by lowering the degree of deacetylation of the chitosan from 90% to 65% (Schipper et al., 1999). This study also used lower Cs concentrations (≤0.1%) which might have mitigated the influence on tight junction integrity. In reducing chitosan concentration from 0.5% to 0.1%, other studies have noted a decrease in paracellular permeability (Artursson et al., 1994; Smith et al., 2004). There is overwhelming evidence of clathrin-mediated endocytosis being responsible for the cellular uptake of chitosan nanoparticles under 200 nm (Ma and Lim, 2003; Zuhorn et al., 2002). Confocal micrographs had revealed the dominance of Cs surrounding the cell surface, while the NP was mostly internalised by the cell (Fig. 4). These micrographs are in agreement with the report from Ma and Lim (2003), which utilised trypan blue to quench the extracellular fluorescence. The selective visualisation method was preferred for this study as the presence of trypan blue tended to complicate sample preparation as well as instrument optimisation to detect the fluorescent FITC signals. Using this technique, FITC-NP was also detected in the cell nucleus whereas nuclear uptake was not evident for the larger-sized insulin-loaded chitosan nanoparticles (504 ± 37 nm) produced by Ma and Lim (2003). Nuclear accumulation of FITC-NP could also disrupt the nucleus and lead to mitochondrial dysfunction, since mitochondrial enzymes, such as mitochondrial DNA polymerase γ are encoded in nuclear genes (Chinnery and Schon, 2003; Van Goethem et al., 2001). TEM micrographs depicted the gross effect on the Caco-2 cell morphology following exposure to 0.1% NP at pH 6.0 for 4 h (Fig. 5). Although an exposure time of 1 h was adequate for the manifestation of any cell membrane damages (Loretz and Bernkop-Schnurch, 2007), the cells were exposed for 4 h to correlate with the MDA and transport experimental data. The disruption of cell structure and nuclear fragmentation observed for the Caco-2 cells was similar to that reported by Qi et al. (2005) for the MGC803 human gastric carcinoma cells. Although Qi et al. (2005) used a 10-fold lower concentration of chitosan nanoparticles, the incubation time was only prolonged to 24 h. The TEM observations were in agreement with the decreases in MDA, which is not surprising given that the rupture of cell membranes is known to be followed by injuries to the mitochondria (Loretz and Bernkop-Schnurch, 2007). Moreover, it distinctly characterises necrosis-mediated cell death from apoptotic cell death (Golstein and Kroemer, 2006; Luciani et al., 2009). Incubation of the cells with 0.005% NP at pH 6.0 did not rupture the cell membrane, although the TEM micrographs did highlight an increase in the number of mitochondrial organelles. Thus, the TEM micrographs may be considered to be generally supportive of the MDA data. Conclusions The data obtained in this study suggest that the in vitro cytotoxicity of chitosan nanoparticles against the Caco-2 cells is less dependent on positive surface charges than on the particle size. Particle size is in turn determined by the pH of the medium in which the nanoparticles are dispersed. At exposure concentration of 0.1%, nanoparticles of 25 nm can be internalised by the Caco-2 cells, and they can proceed to enter the nucleus or inflict extensive damage to the intracellular organelles. Concurrently, the transport of materials along the paracellular pathway is facilitated. The Caco-2 cells are, however, capable of recovering from such assaults

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5 days following NP removal, although a repeat NP exposure will produce similar effects, albeit with comparable cell recovery. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The research and postgraduate studies of JW Loh are supported by grants from the University of Western Australia. The authors thank Dr Johnny Lo (School of Engineering, Edith Cowan University) for his help with the data analyses, the Centre for Strategic Nano-Fabrication, incorporating toxicology, for the use of the SDP and DLS equipment, as well as acknowledge the facilities, scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, the University of Western Australia, a facility funded by the University, State of Western Australia and the Commonwealth Government of Australia. References Ajun, W., Yan, S., Li, G., Huili, L., 2009. Preparation of aspirin and probucol in combination loaded chitosan nanoparticles and in vitro release study. Carbohydr. Polym. 75, 566–574. Artursson, P., Lindmark, T., Davis, S.S., Illum, L., 1994. Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm. Res. 11, 1358–1361. Bejugam, N.K., Sou, M., Uddin, A.N., Gayakwad, S.G., D'Souza, M.J., 2008. Effect of chitosans and other excipients on the permeation of ketotifen, FITC-dextran, and rhodamine 123 through Caco-2 cells. J. Bioact. Compat. Polym. 23, 187–202. Braydich-Stolle, L., Hussain, S., Schlager, J.J., Hofmann, M., 2005. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicol. Sci. 88, 412–419. Chen, F., Zhang, Z.-R., Yuan, F., Qin, X., Wang, M., Huang, Y., 2008. In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery. Int. J. Pharm. 349, 226–233. Cheng, X., Zhang, F., Zhou, G., Gao, S., Dong, L., Jiang, W., Ding, Z., Chen, J., Zhang, J., 2009. DNA/chitosan nanocomplex as a novel drug carrier for doxorubicin. Drug Deliv. 16, 135–144. Chinnery, P.F., Schon, E.A., 2003. Mitochondria. J. Neurol. Neurosurg. Psychiatry 74, 1188–1199. De Angelis, I., Vincentini, O., Brambilla, G., Stammati, A., Zucco, F., 1998. Characterization of furazolidone apical-related effects to human polarized intestinal cells. Toxicol. Appl. Pharmacol. 152, 119–127. de Campos, A.M., Diebold, Y., Carvalho, E.L.S., Sánchez, A., Alonso, M.J., 2004. Chitosan nanoparticles as new ocular drug delivery systems: in vitro stability, in vivo fate, and cellular toxicity. Pharm. Res. 21, 803–810. Dodane, V., Amin Khan, M., Merwin, J.R., 1999. Effect of chitosan on epithelial permeability and structure. Int. J. Pharm. 182, 21–32. Elzatahry, A.A., Mohy-Elidin, M.S., 2008. Preparation and characterization of metronidazole-loaded chitosan nanoparticles for drug delivery application. Polym. Adv. Technol. 19, 1787–1791. Fernández-Urrusuno, R., Calvo, P., Remuñán-López, C., Vila-Jato, J.L., Alonso, M.J., 2004. Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm. Res. 16, 1576–1581. Ferruzza, S., Scarino, M.-L., Rotilio, G., Ciriolo, M.R., Santaroni, P., Muda, A.O., Sambuy, Y., 1999. Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells. Am. J. Physiol. Gastrointest. Liver Physiol. 277, G1138–G1148. Ge, Y., Zhang, Y., He, S., Nie, F., Teng, G., Gu, N., 2009. Fluorescence modified chitosancoated magnetic nanoparticles for high-efficient cellular imaging. Nanoscale Res. Lett. 4, 287–295. Golstein, P., Kroemer, G., 2006. Cell death by necrosis: towards a molecular definition. Trends Biochem. Sci. 32, 37–43. Haas, J., Kumar, M.N.V.R., Borchard, G., Bakowsky, U., Lehr, C.M., 2005. Preparation and characterization of chitosan and trimethyl-chitosanmodified poly-(e-caprolactone) nanoparticles as DNA carriers. AAPS PharmSciTech E22–E30. Hafner, A., Lovric, J., Voinovich, D., Filipovic-Grcic, J., 2009. Melatonin-loaded lecithin/ chitosan nanoparticles: physicochemical characterisation and permeability through Caco-2 cell monolayers. Int. J. Pharm. 381, 205–213. Huang, M., Khor, E., Lim, L.Y., 2004. Uptake and cytotoxicity of chitosan molecules and nanoparticles: effects of molecular weight and degree of deacetylation. Pharm. Res. 21, 344–353. Hurni, M.A., Noach, A.B.J., Blom-Roosemalen, M.C., de Boer, A.G., Nagelkerke, J.F., Breimer, D.D., 1993. Permeability enhancement in Caco-2 cell monolayers by sodium salicylate and sodium taurodihydrofusidate: assessment of effect-reversibility and imaging of transepithelial transport routes by confocal laser scanning microscopy. J. Pharmacol. Exp. Ther. 267, 942–950. Kompella, U.B., Bandi, N., Ayalasomayajula, S.P., 2003. Subconjunctival nano- and microparticles sustain retinal delivery of budesonide, a corticosteroid capable of inhibiting VEGF expression. Investig. Ophthalmol. Vis. Sci. 44, 1192–1201.

282

J.W. Loh et al. / Toxicology and Applied Pharmacology 262 (2012) 273–282

Leonard, M., Creed, E., Brayden, D., Baird, A.W., 2000. Evaluation of the Caco-2 monolayer as a model epithelium for iontophoretic transport. Pharm. Res. 17, 1181–1188. Liang, J., Li, F., Fang, Y., Yang, W., An, X., Zhao, L., Xin, Z., Cao, L., Hu, Q., 2011. Synthesis, characterization and cytotoxicity studies of chitosan-coated tea polyphenols nanoparticles. Colloids Surf., B 82, 297–301. Loh, J.W., Schneider, J., Carter, M., Saunders, M., Lim, L.Y., 2010a. Spinning disc processing technology: potential for large-scale manufacture of chitosan nanoparticles. J. Pharm. Sci. 99, 4326–4336. Loh, J.W., Yeoh, G., Saunders, M., Lim, L.Y., 2010b. Uptake and cytotoxicity of chitosan nanoparticles in human liver cells. Toxicol. Appl. Pharmacol. 249, 148–157. Loretz, B., Bernkop-Schnurch, A., 2006. In vitro evaluation of chitosan-EDTA conjugate polyplexes as a nanoparticulate gene delivery system. AAPS J. 80, E756–E764. Loretz, B., Bernkop-Schnurch, A., 2007. In vitro cytotoxicty testing of non-thiolated and thiolated chitosan nanoparticles for oral gene delivery. Nanotoxicology 1, 139–148. Luciani, M.F., Kubohara, Y., Kikuchi, H., Oshima, Y., Golstein, P., 2009. Autophagic or necrotic cell death triggered by distinct motifs of the differentiation factor DIF-1. Cell Death Differ. 16, 564–570. Ma, Z., Lim, L.Y., 2003. Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles. Pharm. Res. 20, 1812–1819. Madara, J.L., 1998. Regulation of the movement of solutes across tight junctions. Annu. Rev. Physiol. 60, 143. Miret, S., Abrahamse, L., de Groene, E.M., 2004. Comparison of in vitro models for the prediction of compound absorption across the human intestinal mucosa. J. Biomol. Screen. 9, 598–606. Mitra, S., Gaur, U., Ghosh, P.C., Maitra, A.N., 2001. Tumour targeted delivery of encapsulated dextran-doxorubicin conjugate using chitosan nanoparticles as carrier. J. Control. Release 74, 317–323.

Mohy Eldin, M.S., Soliman, E.A., Hashem, A.I., Tamer, T.M., 2008. Antibacterial activity of chitosan chemically modified with new technique. Trends Biomater. Artif. Organs 22, 125–137. Perdikis, D.A., Davies, R., Zhuravkov, A., Brenner, B., Etter, L., Basson, M.D., 1998. Differential effects of mucosal pH on human (Caco-2) intestinal epithelial cell motility, proliferation, and differentiation. Dig. Dis. Sci. 43, 1537–1546. Qi, L.F., Xu, Z.R., Li, Y., Jiang, X., Han, X.Y., 2005. In vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells. World J. Gastroenterol. 11, 5136–5141. Ranaldi, G., Marigliano, I., Vespignani, I., Perozzi, G., Sambuy, Y., 2002. The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line. J. Nutr. Biochem. 13, 157–167. Rodriguez-Juan, C., Perez-Blas, M., Valeri, A.P., Aguilera, N., Arnaiz-Villena, A., PachecoCastro, A., Martin-Villa, J.M., 2001. Cell surface phenotype and cytokine secretion in Caco-2 cell cultures: increased RANTES production and IL-2 transcription upon stimulation with IL-1[beta]. Tissue Cell 33, 570–579. Sambruy, Y., Ferruzza, S., Ranldi, G., de Angelis, I., 2001. Intestinal cell culture models. Cell Biol. Toxicol. 17, 301–317. Schipper, N.G.M., Varum, K.M., Stenberg, P., Ocklind, G., Lennernas, H., Artursson, P., 1999. Chitosans as absorption enhancers of poorly absorbable drugs: 3: influence of mucus on absorption enhancement. Eur. J. Pharm. Sci. 8, 335–343. Smith, J., Wood, E., Dornish, M., 2004. Effect of chitosan on epithelial cell tight junctions. Pharm. Res. 21, 43–49. Van Goethem, G., Dermaut, B., Löfgren, A., Martin, J.-J., Van Broeckhoven, C., 2001. Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat. Genet. 28, 211. Yang, R., Shim, W.-S., Cui, F.-D., Cheng, G., Han, X., Jin, Q.-R., Kim, D.-D., Chung, S.-J., Shim, C.-K., 2009. Enhanced electrostatic interaction between chitosan-modified PLGA nanoparticle and tumor. Int. J. Pharm. 371, 142–147. Zuhorn, I.S., Kalicharan, R., Hoekstra, D., 2002. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J. Biol. Chem. 277, 18021–18028.