Toxicology and Applied Pharmacology 262 (2012) 273–282

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Toxicology and Applied Pharmacology

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

⁎ Corresponding author at: School of Biomedical, Biences, 35 Stirling Hwy, Crawley 6009, Australia. Fax: +

E-mail address: lee.lim@uwa.edu.au (L.-Y. Lim).

0041-008X/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.taap.2012.04.037

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 February 2012Revised 25 April 2012Accepted 30 April 2012Available online 18 May 2012

Keywords:Chitosan nanoparticlesIntestinal cellsNecrosisToxicity

Published toxicology data on chitosan nanoparticles (NP) often lack direct correlation to the in situ size andsurface characteristics of the nanoparticles, and the repeated NP assaults as experienced in chronic use. Theaim of this paper was to breach these gaps. Chitosan nanoparticles synthesized by spinning disc processingwere characterised for size and zeta potential in HBSS and EMEM at pHs 6.0 and 7.4. Cytotoxicity againstthe Caco-2 cells was evaluated by measuring the changes in intracellular mitochondrial dehydrogenase activ-ity, TEER and sodium fluorescein transport data and cell morphology. Cellular uptake of NP was observedunder the confocal microscope. Contrary to established norms, the collective data suggest that the in vitro cy-totoxicity of NP against the Caco-2 cells was less influenced by positive surface charges than by the particlesize. Particle size was in turn determined by the pH of the medium in which the NP was dispersed, withthe mean size ranging from 25 to 333 nm. At exposure concentration of 0.1%, NP of 25±7 nm (zeta potential5.3±2.8 mV) was internalised by the Caco-2 cells, and the particles were observed to inflict extensive dam-age to the intracellular organelles. Concurrently, the transport of materials along the paracellular pathwaywas significantly facilitated. The Caco-2 cells were, however, capable of recovering from such assaults5 days following NP removal, although a repeat NP exposure was observed to produce similar effects to the1st 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 studiedmaterials in the biomedical industry and are highly regarded as a ma-terial of choice in drug delivery applications (Ajun et al., 2009; Chenet al., 2008; Cheng et al., 2009; Fernández-Urrusuno et al., 2004).The objectives are often to reduce drug side effects, control the rateof drug delivery, and ensuring only the targeted area is treated(Elzatahry and Mohy-Elidin, 2008; Mitra et al., 2001). The use of chi-tosan nanoparticles in drug delivery also has the added benefit of in-creasing drug permeation through the absorptive epithelia (Bejugamet al., 2008; Smith et al., 2004).

While the chitosan parent polymer is generally regarded to be safeand biocompatible, it is not unusual to find embedded in these studiesevidence of the cytotoxicity caused by chitosan nanoparticles (Lianget 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 chitosannanoparticles (122±5 nm) exhibited different mechanisms of cellu-lar uptake and distribution in comparison to chitosan, which werepresented as dissolved molecules. Recently, Loh et al. (2010a)

omolecular and Chemical Sci-61 8 6488 7532.

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developed a capacity for synthesising smaller chitosan nanoparticleswith narrow size distribution (18±1 nm) by employing the spinningdisc processing technology (SDP). Subsequent testing of thesenanoparticles indicated that the cell membrane integrity of humanhepatocytes has been compromised, causing necrotic and/or auto-phagic cell death (Loh et al., 2010b). The chitosan nanoparticleshave also caused a dose-dependent increase in CYP3A4 enzyme activ-ity in the human hepatocytes. These results led us to question wheth-er the smaller chitosan nanoparticles would have a similar toxic effecton intestinal cells.

The evaluation of drug toxicity against the intestinalmucosal is impor-tant to the pharmaceutical industry as the oral pathway is the preferredroute for the administration of drugs. The Caco-2 cell line is often usedas the surrogate of the human intestine since it spontaneously differenti-ates after 3–4 weeks into highly polarised enterocyte-like phenotype(Rodriguez-Juan et al., 2001) with functional tight junctions (Leonard etal., 2000) when cultured on porous membranes under normal cultureconditions. The differentiated cells developed well organised microvillion the apical membrane, and expressed many of the enzymes and trans-porters found in absorptive enterocytes (Miret et al., 2004). For these rea-sons, the Caco-2 cell line was selected to investigate the cytotoxicity ofchitosan nanoparticles produced by the SDP.

Apart from nanoparticle cytotoxicity, there are also a number of pub-lished 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(pHb6.5) to facilitate the ionisation of the amino groups in the chitosanpolymer. The resultant positive charges enabled the chitosan nano-particles to associate with the negatively charged sialic acid residues inmucus, thereby imparting a mucoadhesive property (de Campos et al.,2004). However, cell membrane damage could also have resulted fromthe electrostatic interactions between the chitosan nanoparticles andcell membrane, as demonstrated in a study by Loretz and Bernkop-Schnurch (2007), which involved chitosan nanoparticles of 197±54 nmwith 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 en-docytosis has been demonstrated, with twice the amount ofnanoparticles found in the Caco-2 cells after 2 h exposure at 0.1%loading concentration when compared to chitosan molecules (Maand 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 per-meability in a concentration-dependent manner in Caco-2 cell mono-layers. These studies have also demonstrated the reversibility of thetight junctions in Caco-2 cells exposed to a single dosing of chitosannanoparticles. The mechanism appeared to have involved a partialmodulation of the cytoskeleton through induced changes in the cellu-lar 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 havenot provided any clear evidence of the correlation between cells andnanoparticle characteristics. The majority of these studies employedpolydispersed chitosan nanoparticles with mean diameter above 65 nmand size distribution range between 37 nm and 100 nm, making it diffi-cult to attribute the cellular interactions to any specific particle size.More-over, the particle characteristics were not measured in the medium usedfor the cell culture experiments. Therefore, the objectives of this paper are(1) to characterise chitosan nanoparticles in biorelevantmedia to providecloser correlation of their toxicity profile to their in situ characteristics atthe biological interfaces; (2) to evaluate the biological effects of chitosannanoparticles against the Caco-2 cells upon repeat exposure to providea more realistic evaluation of the nanoparticles in clinical use.

Materials and methodology

Materials. Chitosan (medium molecular weight), phosphate bufferedsaline (PBS; 0.138 M NaCl, 0.0027 M KCl; 0.0015 M KH2PO4; 0.0081 MHNa2PO4; pH 7.4), N-acetyl-D-glucosamine, Hank's balanced saltsolution (HBSS), sodium fluorescein (NaF), thiazolyl blue tetrazoliumbromide (MTT), dextran sulphate sodium salt, sodium dodecylsulphate (SDS), isopropanol, 4′,6-Diamidino-2-phenylindole dihydro-chloride (DAPI), glutaraldehyde, tannic acid, Earle's Minimum EssentialMedium (EMEM), non-essential amino acids, penicillin, streptomycinand 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 provid-ed 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, polycarbonatemembrane, New York, USA). Peel-A-Way® embedding moulds werepurchased from Proscitech (22×22 mm, high density polyethylene,Queensland, Australia). All other cell culture plates and flasks werepurchased from Greiner Bio One (Neuburg, Germany).

Test samples. Chitosan nanoparticles were produced by ionotropicgelation using the SDP. Chitosan molecules (Cs) and nanoparticles(NP) were prepared and isolated according to the methods outlinedin 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 (Lohet al., 2010b). Test samples were dispersed in HBSS or supplementedEMEM 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 werecharacterised by shape, size and zeta potential using dynamic lightscattering (DLS) and laser Doppler anemometry techniques, respec-tively (Zetasizer Nano ZS, Malvern Instruments, Version 4, Worcs.,UK).

Cell culture. Caco-2 cells (passage 40) purchased from the EuropeanCollection of Cell Cultures (ECACC, Salisbury, Great Britain) weregrown in 75 cm2 culture flasks at 37 °C in 5% CO2 and 90% relative hu-midity (Thermo forma series 2, Ohio, USA) in 10 mL of EMEM sup-plemented with 1% non-essential amino acids, 10,000 U/mLpenicillin, 10 mg/mL streptomycin, 200 mM glutamine and 10% foetalbovine serum. Spent culture medium was exchanged every 2–3 dayswith 10 mL of fresh medium and sub-cultured at >80% confluencyfor experiments. Cells between passages 46 and 66 were used for sub-sequent experiments.

Mitochondrial dehydrogenase activity (MDA). The analysis of MDAprovides an overview of the cytotoxicity inflicted by the test samples.In this study, MDA analysis was also used to determine the length ofexposure time (h) for subsequent experiments. Caco-2 cells wereplated in 96-well plates at 10,000 cells per well and cultured over48 h with 100 μL of supplemented EMEM. The spent medium was as-pirated and the cells were incubated with samples at pH 7.4 or pH 6.0for 4–72 h at 37 °C in 5% CO2 and 90% relative humidity. Experimentsup to 24 h incubation used HBSS as the vehicle. Experiments involv-ing 48 or 72 h exposure used culture medium as the vehicle, as thecells could not survive for long periods in HBSS. For the 72 h exposureexperiments, the samples were removed at 48 h, the cells werewashed once with pre-warmed culture medium (100 μL) and incu-bated with corresponding fresh Cs or NP samples for a further 24 h.After the specified exposure times, the cells were washed once with100 μL of pre-warmed PBS before they were incubated for 2 h with100 μL of MTT solution (1 mg/mL in PBS). Following the aspirationof the MTT solution, the cells were solubilised with 100 μL of 10% w/vSDS (0.01 M HCl in isopropanol as vehicle) overnight to extract theintracellular formazan crystals. The colour of the cell lysate wasmeasured at 570 nm (BioTek EL808, Vermont, USA) and the resultswere expressed as a percentage activity relative to cells exposed tothe corresponding blank vehicles. Dextran sulphate sodium salt(0.1% w/v) and SDS (0.1% w/v) in corresponding vehicles were usedas positive and negative controls, respectively.

Transport studies. Transport studies examine the capacity of chitosanto modulate the tight junctions in differentiated Caco-2 cells. The cellsat a seeding density of 10,000 cells/well were grown on the apicalchamber of 24-well Transwell® permeable supports in 0.1 mL ofpre-warmed EMEM with 0.6 mL of EMEM in the basal compartment.Spent medium was exchanged every other day. After 25 days of cul-ture, the cells were prepared for transport experiments by washingonce with pre-warmed HBSS (pH 6.0), and equilibrated with pre-warmed HBSS (0.1 mL apical, 0.6 mL basal) for 30 min in the CO2 in-cubator. The integrity of the cell monolayers was monitored throughthe measurement of transepithelial electrical resistance (TEER) usingthe MilliCell®-ERS Chopstick Electrodes (Millipore, Massachusetts,USA). Cells with a net TEER ≥200 Ω.cm2 were used for subsequenttransport experiments.

Samples of Cs or NP were prepared in pH 6.0 HBSS medium con-taining 0.01% of sodium fluorescein (NaF, 37.6 M). The transport me-dium (HBSS containing 0.01% w/v NaF) served as the negativecontrol. To start the transport experiments, the medium in the basalchamber was replaced with fresh pre-warmed HBSS while that inthe apical chamber was replaced with 0.1 mL of pre-warmed samples.

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

Media Chitosan Nanoparticles

pH 7.4 pH 6.0 pH 7.4 pH 6.0

Particle size (nm)HBSS 1817±309 1809±171 333±43 25±7EMEM * 1558±301 149±16 26±2

Zeta potential (mV)HBSS −5.4±0.9 9.9±1.9 3.3±0.4 5.3±2.8EMEM −8.5±0.8 8.8±1.0 −1.4±0.2 −6.1±1.9

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After predetermined periods (30, 60, 120 and 180 min) of incubationat 37 °C in 5% CO2/95% relative humidity, 200 μL of medium in thebasal compartment was withdrawn for NaF analysis at 490 nm. Thewithdrawn aliquot was immediately replaced with an equal volumeof fresh pre-warmed HBSS to maintain media volume in the basalchamber. At the final time point of sampling, the NaF content in200 μL of medium from the apical compartment was also measured.

Immediately after the last time point of sampling, the cells werewashed once with HBSS before they were returned to the CO2 incuba-tor with pre-warmed EMEM. During this recovery period, the spentmedium was exchanged once. On the 5th day, the tight junction in-tegrity of the cell monolayers was assessed in the transport medium.The next day, the cells were subjected to a second cycle of exposure tothe Cs and NP samples followed by a recovery period using the sameexperimental conditions as described for the first cycle.

Laser scanning confocal microscopy. Cs and NP uptakes into Caco-2cell monolayers were evaluated using laser scanning confocal micros-copy. Caco-2 cells (2×105) were seeded onto a glass cover (#1 thick-ness) placed in a 22-mm Petri dish with 1.5 mL of EMEM. At fullconfluency (48 h), the spent medium was replaced with 1.5 mL ofFITC-Cs or FITC-NP dispersion (0.1% w/v in pH 6.0 HBSS). After 4 hof incubation, the cells were washed twice with PBS, fixed in parafor-maldehyde (4% w/v in PBS) for 15 min, and then washed thrice withPBS. Viable cells were differentiated by nuclear staining with DAPI(2 mM in PBS) for 10 min, the excess stain removed by washingwith PBS for 1 min before the cover slip was mounted onto a cleanslide for viewing under the laser scanning confocal microscope(Leica TCS SP2, Illinois, USA). FITC-Cs and FITC-NP were detected atλex: 488 nm, λem: 536–624 nm while the DAPI fluorescence wasdetected at a lower λex: 385 nm and λem: 400–480 nm.

Transmission electron microscopy (TEM). TEM is used to assess themorphology of Caco-2 cell monolayers following exposure to thetest samples. Caco-2 cells (200,000 cells) were seeded on Peel-A-Way® embedding moulds with 1.5 mL of culture medium andgrown for 4 days before incubation with 1 mL of the test samples intransport medium. HBSS at pH 6.0 served as the transport mediumas well as the negative control. After 4 h, the samples were removedand the cells were washed once with 1.5 mL of PBS before overnightincubation at ambient temperature with 1.5 mL of prefix solution(2.5% glutaraldehyde and 1% tannic acid in PBS). Then the cells werestained with 1% osmium in PBS and embedded in Spurrs® resin forviewing under the TEM (JOEL JEM 2100, Tokyo, Japan) at 120 kVwith a spot size of 1 and alpha 3.

Statistical analysis. Data are expressed as mean±standard deviationobtained from ≥3 independent experiments conducted on differentdays with ≥1 repeat wells per experiment. Statistical analysis ofdata was performed using analysis of variance (ANOVA) with post-hoc Tukey's test for paired comparison of the means (SPSS Version11, Lead Technologies Inc., Chicago, USA).

Results

Test sample properties

Cs and NP characteristics differed in the two media and at the twopHs used (Table 1). NP particle size as measured by the DLS was about6 to 13 fold larger at pH 7.4 than at pH 6.0, indicating significant par-ticle aggregation at neutral pH, particularly in the HBSS medium. AtpH 7.4, there was a 2-fold increase in particle size when the mediumwas switched from EMEM to HBSS, whereas comparable NP particlesizes were observed at pH 6 in both media. These changes in particlesize did not correlate well with the zeta potential values measured inthe respective media. While relatively low zeta potential values were

registered for the NP in both media, there were clear trends of posi-tive values in HBSS and negative values in EMEM, with an increasein zeta potential magnitude upon acidification of the respectivemedia to pH 6.0.

Cs particle size was much higher and consistently above 1 μm atboth pHs in the two media. Unlike the NP particles, the zeta potentialof the Cs particles was less affected by the composition of the bio-relevant medium than by their pH, exhibiting negative charges atpH 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 and72 h exposure at pH 6.0 and pH 7.4 (Fig. 1). As with many publishedin vitro cytotoxicity studies, to ensure cell survival, the experimentswere 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 isthe relative innocuity of the Cs samples at the range of concentrationsand time points tested. Except for cells exposed for 48 h to 0.1% Cs inEMEM (Fig. 1, 3B), which had cell viability slightly below 80%, theCaco-2 cells were observed to be generally resilient against Csexposure.

Unlike the Cs samples, the NP samples showed low cytotoxicityprofiles only at pH 7.4. At this pH, for all concentration ranges andtime points tested, the NP did not lower the viability of the Caco-2cells to below 80%, except for 0.1% NP at 4 h exposure, where cell vi-ability of about 60% was obtained (Fig. 1, 1A). On the other hand,when the experimental pH was lowered to 6.0, cell viability below40% was observed for 0.1% NP at all exposure times evaluated, aswell 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 posi-tive zeta potential of 5.3 mV, while the Cs sample had mean size of1809 nm and zeta potential of 9.9 mV. Tight junction permeabilityin 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 baselineTEER (measured just prior to experiment initiation) and plottedagainst exposure time. Over a 3 h period of exposure to Cs, the TEERvalues of the Caco-2 cell monolayers remained above 84% of the base-line value, similar to those observed in cells exposed to HBSS (Fig. 2IA). Exposure to a second sample of Cs, following 5 days of recoveryin EMEM after the 1st Cs exposure, produced a similar TEER profilein the Caco-2 cells (Fig. 2 IIA), the TEER values remaining above 83%at all concentrations studied. The retention of tight junction integrityduring both Cs exposures and the respective recovery periods wasconfirmed by the fluorescein transport data (Fig. 3A). The Papp values

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 mediumas 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 de-viation, n=3. a denotes significant difference to dextran (negative control) and b denotes significant difference to chitosan at pb0.05.

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obtained were comparable to those for the negative control cells evenat the highest applied concentration of 0.10% Cs (p>0.05).

On the other hand, the NP samples led to concentration-dependent decreases in TEER within 0.5 h of exposure, with TEER fall-ing 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 withthe TEER values recorded at 24%, 5% and 3%, for the respectiveconcentrations at 3 h. Despite the sharp fall in TEER values, acorresponding increase in fluorescein transport was observed onlyat the highest NP concentration of 0.1% (Fig. 3B). It is not unusualthat the trends in TEER and Papp values did not develop in parallelas they reflected different functional properties (Madara, 1998).Mean fluorescein Papp, measured after 3 h of transport in the presenceof 0.1% of NP was significantly higher (7.5±1.6×10−7 cm/s) than thevalue obtained for control cells (2.5±1.2×10−7 cm/s; p=0.023).

However, the NP-induced changes in TEER and fluorescein Pappwere temporary, even for those exposed to 0.1% of NP. The cell mono-layers, after 5 days of incubation with EMEM post-NP exposure, werefound to exhibit TEER comparable to those measured in the negativecontrol cells (Fig. 2 ID). This recovery in tight junction integrity wassupported by fluorescein Papp values (Fig. 3B), which were no differ-ent from those obtained in control cells.

The 2nd dose of NP caused the TEER to fall lower than that mea-sured 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. Itfell further to b6% of baseline TEER after 3 h at all 3 NP concentra-tions. Once more, the fluorescein transport data did not reflect thedramatic drop in TEER, as the fluorescein Papp values obtained forthe 2nd dose of NP were similar to those recorded for the 1st NPdose (Fig. 3B). Similarly, only those cells exposed to 0.1% of NP

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 fluo-rescein 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 mon-itoring 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 experimentcommencement.

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showed significantly higher Papp, at 6.4±1.3 (×10−7 cm/s), than thenegative control cells.

Remarkably, the changes in cell monolayer integrity associatedwiththe 2nd dose of NP were also reversible following 5 days of incubation

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

Fig. 3. The apparent permeability coefficient (Papp) values obtained for sodium fluores-cein transport. Dose 1 represents cells exposed to the 1st cycle of Cs (A) or NP (B) at pH6.0. Recovery 1 represents data obtained from the same cells grown in culture mediumfor 5 days following 1st exposure. Dose 2 represents data obtained from the cells ex-posed to the 2nd cycle of Cs or NP after the recovery period. Recovery 2 representsdata obtained from cells grown in culture medium for 5 days following 2nd exposure.Results are expressed as mean±standard deviation (n≥3); * indicates statistical dif-ference compared to HBSS.

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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 cellmonolayers 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 theCaco-2 cell monolayers grown on the cover slips were approximately10 μm in thickness. To differentiate between particles attached on thesurface of the cell and those internalised into the cells, micrographs ofthe cell surface and at a depth of 5 μm from the cell surface weretaken.

Micrographs of the cell surface showed strong fluorescent signalsfor cells exposed to FITC-Cs (Fig. 4A) whereas only faint fluorescencewas detected for cells exposed to FITC-NP (Fig. 4B). In contrast, whenthe analysis was focused at a depth of 5 μm from the cell monolayersurfaces, minimal fluorescence was detected for cells exposed toFITC-Cs (Fig. 4C) while intense fluorescent signals were detected forthe FITC-NP-exposed cell samples (Fig. 4D). Staining of the cellnucleic acids with DAPI allowed for visualisation of the cell nucleusat a lower wavelength (λex: 385 nm and λem: 400–480 nm) thanthat for the FITC. An overlay of the images produced from the DAPI(blue) and FITC (green) signals (Fig. 4E) showed extensive co-localisation of the DAPI and FITC, confirming the presence of NP inthe nucleus of the Caco-2 cells.

Transmission electron micrographs

TEMwas used to detect changes in the cell ultrastructures after in-cubation 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.

Figs. 5G and H showed extensive fragmentation of intracellular struc-tures within the Caco-2 cells, accompanied by the condensation of thecell nucleus following exposure to 0.1% of NP. In contrast, the sameconcentration of Cs did not result in significant changes to the cellmorphology (Figs. 5C and D) compared to the control cells (Figs. 5Aand B). Salient subcellular structures, such as the mitochondria,could be clearly delineated in the micrographs. Exposure to a muchlower NP concentration of 0.005% did not result in the extensive dam-age evident in cells exposed to 0.1% NP. However, there was an appar-ent increase in the number of mitochondrial organelles, particularlyin close proximity to the cell nucleus, in cells exposed to 0.005% ofNP (Figs. 5E and F).

Discussion

The results of this study showed that the Caco-2 cells were moresensitive to the cytotoxicity of NP at pH 6.0 than at pH 7.4 for allthe exposure times assessed by MDA (Fig. 1). The same phenomenonwas observed for chitosan molecules and nanoparticles in a numberof studies (Loretz and Bernkop-Schnurch, 2007; Mohy Eldin et al.,2008; Yang et al., 2009), and was often attributed to chitosanpossessing higher positive surface charges due to more extensive pro-tonation of its amino groups at pH below its pKa. This explanation is,however, disputable in the present study, as the zeta potentials mea-sured in HBSS between pH 6.0 and pH 7.4 were not significantly dif-ferent for the NP sample (Table 1). In addition, cytotoxicity wasobserved for the 0.1% NP sample dispersed in EMEM (48 and 72 h ex-posure) at pH 6.0, when the NP possessed negative zeta potentials.The collective data suggest that the size of NP is a more plausible ex-planation 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 theshort term as Caco-2 cells had been demonstrated to withstand pHas low as 5.7 for up to 2 h (Loretz and Bernkop-Schnurch, 2007).However, the motility and proliferation of Caco-2 cells were reportedto have decreased by as much as 31.4% after 48 h following a changein culture medium pH from 7.4 to 7.0 (Perdikis et al., 1998). A similarpredisposition to optimal growth and function at pH 7.4 might existfor the Caco-2 cells in this study, as it was observed that cells exposedfor >4 h to blank pH 6.0 media, be it HBSS or culture medium, were ofa lighter colour compared to cells exposed to corresponding media atpH 7.4. The cells at pH 6.0 might therefore be more susceptible to theeffects of NP, although, based on the MDA, they remained resilient tothe presence of Cs, even after 72 h of co-incubation.

The low cytotoxicity of Cs could not be attributed to a lack of pos-itive charges on the molecules, as the Cs samples exhibited positivezeta potentials, which reached almost +10 mV, in the HBSS andEMEM media at pH 6.0. More likely, the relative lack of cytotoxicitywas associated with the micron size particles obtained when Cs wasdispersed in either media at the two pHs used. Micron sized particlesare known to be effectively excluded from cellular uptake into the cy-toplasm, and this was confirmed by electron micrographs in thisstudy.

In agreement with previous studies by our laboratory employinglarger chitosan nanoparticles (110 nm – 390 nm) (Huang et al.,2004; Ma and Lim, 2003), the poorer cellular internalisation of Csmight account for the different cellular responses of the Caco-2 cellstowards the Cs and NP samples. Unlike the NP, which was shown byconfocal microscopy to be extensively internalised by the cells, Cswas poorly taken up into the cytoplasm despite strong adherence tothe cell surface. The poor cellular internalisation of Cs was likelydue to its large size (≫1 μm) in the two media. It is interesting tonote that, while the current study showed that Cs at up to 0.1% didnot affect the viability of the Caco-2 cells as measured by MDA, it

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.

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was shown to promote cytoproliferation of the A549 cells in an earlierstudy 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 pro-longed chitosan exposures, a hallmark of chronic therapy wheredrugs are administered in repeat doses. In this study, the MDA datasuggests that a prolongation of NP exposure to 72 h did not produceadditional toxicity to the Caco-2 cells at pH 6.0, particularly whenthe NP loading concentration was not more than 0.05%. In fact thereappears to be a recovery of cell viability for the 0.025% NP sample inHBSS when the exposure time was prolonged from 4 to 24 h(Figs. 1, 1B vs 2B). These results are not in agreement with the find-ings of Qi et al. (2005), who observed chitosan nanoparticles to bemore cytotoxic and exhibited a 3-fold reduction in IC50 value from16.2 to 5.3 μg/mL when the incubation time was extended from 24to 48 h in the MGC803 cells. Underlying reasons could be the differ-ences in cell type, as well as the nanoparticle characteristics, for thechitosan nanoparticles prepared by Qi et al. (2005) had larger

diameter of 65 nm. Another reason might be that the Caco-2 cellsresponded to the NP assault by increasing intracellular mitochondriaexpression, as was observed in the TEM micrographs of cells exposedto 0.005% NP (Figs. 5E and F). However, as the concentration of0.005% was substantially lower than the concentrations of NP usedin the MDA assessment, further confirmatory experiments are re-quired, since there is also no published report correlating internalisedchitosan nanoparticles to the promotion of mitochondrial expressionin cell cultures.

Published paracellular transport studies involving chitosannanoparticles 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 inuse. Our data showed that, following co-incubation with the firstdose of NP at 0.1%, the paracellular transport of sodium fluoresceinwas significantly facilitated, but the effect was reversible withindays of removing the NP. This observation was consistent with pub-lished data, one of which showed TEER values to be restored 14 hafter the removal of chitosan nanoparticles (Dodane et al., 1999;

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.

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Loretz and Bernkop-Schnurch, 2007). We have, however, evaluatedthe effects of a second NP exposure and the subsequent cell recovery.The stronger cellular response to the 2nd dose of NP, based on theTEER data, implies that the cells had only partially recovered fromthe assault of the 1st NP dose. This is unsurprising given that the de-gree of reversibility of cellular response is inversely proportional tothe 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 thatthe TEER of the Caco-2 cell monolayers was brought below 10% ofbaseline levels by the second NP assault.

The integrity of the tight junctions, as represented by the TEERprofiles, is modulated by cytoskeletal proteins such as actin and tubu-lin 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 byredistributing such proteins to the cytoskeleton (Smith et al., 2004).Data from this study did not mirror this effect, as the paracellularfluorescein transport profiles of Cs-exposed cells were similar to thecontrol cells (Fig. 3A). The anomaly might have arise from the lowerdegree of deacetylation of Cs used in this study (79±2%) comparedto the chitosans reported in the literature (>85%) (Artursson et al.,1994; Schipper et al., 1999; Smith et al., 2004). It was reported thatthe paracellular permeability can be reduced from 1.2×10−6 cm/sto as much as 5×10−7 cm/s by lowering the degree of deacetylationof the chitosan from 90% to 65% (Schipper et al., 1999). This study alsoused lower Cs concentrations (≤0.1%) which might have mitigatedthe influence on tight junction integrity. In reducing chitosan concen-tration from 0.5% to 0.1%, other studies have noted a decrease in para-cellular permeability (Artursson et al., 1994; Smith et al., 2004).

There is overwhelming evidence of clathrin-mediated endocytosisbeing responsible for the cellular uptake of chitosan nanoparticlesunder 200 nm (Ma and Lim, 2003; Zuhorn et al., 2002). Confocal mi-crographs had revealed the dominance of Cs surrounding the cell sur-face, while the NP was mostly internalised by the cell (Fig. 4). Thesemicrographs are in agreement with the report from Ma and Lim(2003), which utilised trypan blue to quench the extracellular fluo-rescence. The selective visualisation method was preferred for thisstudy as the presence of trypan blue tended to complicate samplepreparation as well as instrument optimisation to detect the fluores-cent FITC signals. Using this technique, FITC-NP was also detected inthe cell nucleus whereas nuclear uptake was not evident for thelarger-sized insulin-loaded chitosan nanoparticles (504±37 nm)produced by Ma and Lim (2003). Nuclear accumulation of FITC-NPcould also disrupt the nucleus and lead to mitochondrial dysfunction,since mitochondrial enzymes, such as mitochondrial DNA polymeraseγ are encoded in nuclear genes (Chinnery and Schon, 2003; VanGoethem et al., 2001).

TEMmicrographs depicted the gross effect on the Caco-2 cell mor-phology following exposure to 0.1% NP at pH 6.0 for 4 h (Fig. 5). Al-though an exposure time of 1 h was adequate for the manifestationof any cell membrane damages (Loretz and Bernkop-Schnurch,2007), the cells were exposed for 4 h to correlate with the MDA andtransport experimental data. The disruption of cell structure and nu-clear fragmentation observed for the Caco-2 cells was similar to thatreported by Qi et al. (2005) for the MGC803 human gastric carcinomacells. Although Qi et al. (2005) used a 10-fold lower concentration ofchitosan nanoparticles, the incubation time was only prolonged to24 h. The TEM observations were in agreement with the decreasesin MDA, which is not surprising given that the rupture of cell mem-branes is known to be followed by injuries to the mitochondria(Loretz and Bernkop-Schnurch, 2007). Moreover, it distinctlycharacterises necrosis-mediated cell death from apoptotic cell death(Golstein and Kroemer, 2006; Luciani et al., 2009). Incubation of thecells with 0.005% NP at pH 6.0 did not rupture the cell membrane, al-though the TEM micrographs did highlight an increase in the numberof mitochondrial organelles. Thus, the TEM micrographs may be con-sidered to be generally supportive of the MDA data.

Conclusions

The data obtained in this study suggest that the in vitro cytotoxicity ofchitosan nanoparticles against the Caco-2 cells is less dependent on posi-tive surface charges than on the particle size. Particle size is in turn deter-mined by the pHof themedium inwhich the nanoparticles are dispersed.At exposure concentration of 0.1%, nanoparticles of 25 nm can be inter-nalised by the Caco-2 cells, and they can proceed to enter the nucleus orinflict 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

5 days following NP removal, although a repeat NP exposurewill producesimilar 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 supportedby grants from the University of Western Australia. The authors thankDr Johnny Lo (School of Engineering, Edith Cowan University) for hishelp 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 assistanceof the Australian Microscopy & Microanalysis Research Facility at theCentre for Microscopy, Characterisation & Analysis, the University ofWestern Australia, a facility funded by the University, State of WesternAustralia and the Commonwealth Government of Australia.

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