effect of n-betainate and n-piperazine derivatives of chitosan on the paracellular transport of...

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 226–234

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Effect of N-betainate and N-piperazine derivatives ofchitosan on the paracellular transport of mannitolin Caco-2 cells

Timo Korjamoa,d,∗, Jukka Holappab,c, Sanna Taimistoa, Jouko Savolainenc,Tomi Järvinenb, Jukka Mönkkönena

a Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finlandb Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finlandc Fennopharma Ltd., Microkatu 1, FI-70210 Kuopio, Finlandd Novamass Ltd., Kiviharjuntie 11, FI-90220 Oulu, Finland

a r t i c l e i n f o

Article history:

Received 12 February 2008

Received in revised form 6 July 2008

Accepted 7 July 2008

Published on line 15 July 2008

Keywords:

Absorption enhancer

Caco-2 cells

Chitosan

a b s t r a c t

The effects of novel quaternary chitosan derivatives on the paracellular transport of man-

nitol and cell viability were studied in the Caco-2 cell model. The N-betainate derivative

with the degree of substitution of 0.05 was very effective at 1.0% (w/v) concentration. The

activity decreased as the degree of substitution increased. The cytotoxicity of N-betainates

was rather low. The N-piperazines were at least equally effective as the N-betainates with a

similar degree of substitution (>0.15). Most of the N-piperazines did not exert toxic effects

on the cell monolayers. Overall, the inverse proportionality between the degree of substitu-

tion and activity suggests that an intact chitosan backbone is essential for the bioactivity of

chitosan derivatives. The quaternary group does not substitute for the activity of the free

amine group. In particular, the N-betainate derivatives of chitosan should contain only the

Cytotoxicity minimum number of substituents required for water solubility.

© 2008 Elsevier B.V. All rights reserved.
Permeation
Paracellular transport

1. Introduction

Chitosan (poly-1,4-�-d-glucosamine) is a cationic biopoly-mer that is industrially prepared from chitin. Chitosan hasattracted considerable attention in the pharmaceutical andbiomedical fields not only because of its unique activity prop-erties, but also due to its biocompatibility, biodegradability
and mucoadhesivity. The ability of chitosan to enhance gas-trointestinal drug absorption has been of special interest.Illum et al. (1994) proposed that the mechanism of absorp-tion enhancement was a combination of mucoadhesion and
∗ Corresponding author at: Department of Pharmaceutics, University oTel.: +358 17 162429; fax: +358 17 162252.

E-mail address: [email protected] (T. Korjamo).0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2008.07.001

a loosening effect on the tension of the tight junctions thatexist between cells and this hypothesis has been confirmedby subsequent studies. Chitosan can enhance the paracellu-lar permeation by mediating a structural reorganization of thetight junction proteins ZO-1 and occludin (Dodane et al., 1999;Smith et al., 2004, 2005). This enhancement of paracellulartransport is reversible as well as being time and dose depen-

f Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland.

dent (Dodane et al., 1999). It is also dependent on the molecularweight and the degree of N-acetylation of chitosan (Schipperet al., 1996). Lehr et al. (1992) were the first to describe themucoadhesive properties of chitosan in vitro. The positively

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dx.doi.org/10.1016/j.ejps.2008.07.001

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 226–234 227

Table 1 – N-Betainate derivatives of chitosan

Derivative Degree of substitution Mn Mw Structure

1a 0.05 27,600 42,1002a 0.15 24,800 48,0003a 0.4 11,800 25,0004a 0.6 20,800 48,5005a 0.6 38,100 137,0006a 0.8 19,500 45,3007a 0.8 52,600 73,6008a 0.9 27,200 71,400

9a Glucosamine N-betainate (monomer)

charged chitosan can bind to the negatively charged materi-als of cell surfaces and mucus, e.g., mucins and sialic acids(Qaqish and Amiji, 1999).

Quaternary ammonium derivatives of chitosan have inter-esting pharmaceutical applications, since it is the polycationicproperties of chitosan that are thought to be responsible for

most of the desired activities. These derivatives have twomajor advantages over the parent chitosan: (1) they are water-soluble at physiological pH and (2) they possess a permanentpositive charge on the polysaccharide backbone. The mostextensively evaluated quaternary chitosan derivative is N,N,N-trimethyl chitosan chloride (TMC). Various studies have been

Table 2 – N-Piperazine derivatives of chitosan

Derivative Degree of substitution Mn Mw Structure

1b 0.15 23,900 48,0002b 0.42 17,400 34,5003b 0.61 15,040 15,7004b 0.87 17,200 28,600

5b 0.4 30,200 67,4006b 0.46 20,400 39,2007b 0.56 21,900 23,3008b 0.85 21,800 34,200

9b 0.34 17,600 31,40010b 0.54 17,000 26,30011b 0.56 32,100 33,50012b 0.65 11,200 17,500

13b 0.3 24,700 42,90014b 0.6 16,600 35,00015b 0.9 24,200 49,400

228 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 226–234

carried out on different aspects of the synthetic preparation ofTMC (Sieval et al., 1998; Hamman and Kotze, 2001; Curti et al.,2003; Polnok et al., 2004). However, the synthetic preparation ofTMC is somewhat problematic, i.e., TMC cannot be preparedwithout also methylating the hydroxyl moieties in chitosan(Sieval et al., 1998). For example, higher degrees of quater-nization cannot be prepared without total O-methylation ofthe material (Sieval et al., 1998).

N,N,N-Trimethyl chitosan chlorides have been used in avariety of pharmaceutical applications (Van der Merwe et al.,2004). However, none of the published articles have definedthe degree of O-methylation of the material, although thepresence of O-methyl groups has been well documented asdiscussed above. This information is crucial if one wishes tocharacterize in detail the structure-activity relationships indifferent applications.

In the present study, two types of water-soluble qua-ternary chitosan derivatives were studied, i.e., (1) chitosanN-betainates (compounds 1a–8a) and (2) mono- and diquater-nary piperazine derivatives (compounds 1b–12b) of chitosan.These compounds can be prepared such that they possessvarious degrees of quaternization and without any side-reactions (Holappa et al., 2004, 2006a). The only drawbackencountered in the synthetic routes was the somewhat dras-tic molecular weight reduction of the polymer (Holappa etal., 2004, 2006a). With these well-defined chitosan deriva-tives, it is possible to determine with great accuracy theeffect of the degree of quaternization on the extent of absorp-tion enhancement. These quaternary chitosan derivativesare extremely water-soluble, e.g., 4–6% (w/v) aqueous solu-tion could be prepared at pH 7, while unmodified chitosanis practically insoluble under neutral conditions (Holappa etal., 2004, 2006a). Tertiary N-piperazine derivatives of chitosan(13b–15b) and glucosamine N-betainate (9a) were also studied.The effect of these novel chitosan derivatives on paracellu-lar permeation was studied at neutral pH with [3H]-mannitolas the marker molecule. Possible cytotoxicity was evaluatedwith the lactate dehydrogenase (LDH) release test using thesame filter-grown cells that were used in the permeationexperiments.

2. Material and methods

2.1. Chitosan and chitosan derivatives

ChitoClear chitosan from Primex ehf (Reykjavik, Iceland) withMw of 201 kDa and Mn of 89.8 kDa was used. The degree ofdeacetylation of the material was 85%.

Quaternary chitosan N-betainates (Holappa et al., 2004),quaternary N-piperazine derivatives of chitosan (Holappa etal., 2006a), tertiary piperazine derivatives of chitosan (Holappaet al., 2005) and glucosamine N-betainate (Holappa et al.,2006b) were synthesized as previously described. The chemi-cal structures, degrees of substitution and molecular weightsare shown in Tables 1 and 2. The molecular weights of thestarting material and products were determined by GPS-LSand the degrees of substitution were determined by 1H NMR.Chitosan derivatives were dissolved into HBSS–Hepes at con-centrations three, 0.25, 0.5 and 1.0% (w/v).

2.2. Cell culture and permeation experiments

Caco-2 cell (ATCC HTB-37, Manassas, VA) cell lines werecultured and permeation experiments were performed as pre-viously described (Korjamo et al., 2005). Briefly, monolayersgrown on polycarbonate filters (pore size 0.4 �M, area 1.1 cm2,Costar, Corning, NY) were washed twice with HBSS–Hepesbuffer (pH 7.4) and equilibrated for 15–30 min. TEER val-ues were recorded (Epithelial VoltOhmMeter, World PrecisionInstruments, Sarasota, FL) and only monolayers with TEERvalues >300 � cm2 were accepted for the permeation exper-iments. A total of 500 �l of buffer (2 �l/ml of [3H]-mannitol,specific activity 1 mCi/ml, PerkinElmer, Boston, MA) contain-ing either chitosan derivatives at different concentrationsor control substances (blank, 1% Triton X-100, 2.5 mM EDTAor 0.5% unmodified chitosan) was applied onto the cellmonolayers to initiate the study. Samples from the baso-lateral chamber were withdrawn at regular intervals up to120 min and replaced with fresh buffer. Samples were ana-lyzed with MicroBeta liquid scintillation counter (Wallac,Finland) using OptiPhase HiSafe scintillation cocktail (Wallac,Finland). Since the flux of mannitol across Caco-2 monolayerwas not constant in many chitosan wells, no definite appar-ent permeability coefficient could be calculated. Therefore,the cumulative amount of mannitol transport (% of the initialdonor) at the end of the experiment was used to compare thederivatives. The flux of mannitol was constant in the buffercontrols and the apparent permeability could be calculated inthese wells using the traditional equation J = Papp × A × C0.

2.3. Cytotoxicity assay

The possible cytotoxicity was measured with the LDH releasetest (non-radioactive cytotoxicity assay kit, Promega, Madison,WI). Samples from the apical compartment were withdrawn atthe end of the permeation experiment and diluted appropri-ately. A standard curve (6.5–205 U/l) was diluted from an LDHsuspension (Fluka, Germany). A total of 50 �l of diluted sam-ple or standard was pipetted to a 96 well plate well and then50 �l of LDH substrate was added and the plate was incubatedat +37 ◦C for 30 min while protected from light. The reactionwas stopped by adding 50 �l of stop solution. Absorbance wasrecorded at 490 nm (VICTOR2, Wallac, Finland). Optical den-sities were background corrected and the LDH activity wasdetermined using the standard curve. The LDH release wasscaled to the maximal LDH release (Triton X-100 wells) byequation:

LDHsample − background

LDHTriton X-100 − background× 100%

2.4. Statistical analysis

Both derivative groups (N-betainates and N-piperazines) weretreated separately. Non-parametric Kruskall–Wallis test wasused to detect statistical differences between groups. Individ-ual values were compared pair-wise to the buffer control withDunn’s post hoc test. All statistical analyses were performedwith Prism 4.03 software (GraphPad, San Diego, CA).


Fig. 1 – The effect of the degree of substitution of chitosanderivatives (1.0% m/v concentration) on the enhancement ofparacellular transport in Caco-2 cells. Solidsquares = N-betainates, open diamonds = monoquaternaryN-piperazines with the charge on the outer nitrogen,squares = monoquaternary N-piperazines with the chargeon the inner nitrogen, triangles = diquaternaryNl

3

3

TcTtdpc

Fig. 2 – The time course of mannitol transport acrossCaco-2 monolayer in the presence of buffer control (solidsquares), 0.5% chitosan (solid triangles), 1.0% N-betainatederivative 1a (open diamonds) and 1.0% N-piperazinederivative 7b (open triangles). Mannitol and chitosanderivatives were applied to the pre-equilibrated monolayersat 0 min. The mannitol flux to the basolateral chamber wasfollowed for up to 120 min. Symbols represent average and
-piperazines and crosses = tertiary N-piperazines. Dashed
ine shows the transport of mannitol in control wells.

. Results

.1. Permeation

he effects of N-betainate and N-piperazine derivatives ofhitosan on the transport of mannitol are presented inables 3 and 4. Fig. 1 shows the correlation between manni-
ol transport and the degree of substitution. The N-betainateerivative with the lowest degree of substitution (0.05, com-ound 1a) was the most potent in this group. However, alear effect was observed only at 1% concentration. The
Table 3 – The effect of N-betainate derivatives of chitosan on th

Controls Mean

Buffer (pH 7.4) 2.Buffer (pH 5.5) 2.EDTA 2.5 mM 1.Triton X-100 55.

0.25%

Mean (%) S.D. (%) Mea

Chitosan (pH 7.4) 3Chitosan (pH 5.5) 61a 2.9 0.7 32a 2.6 0.4 23a 2.9 1.1 24a 2.5 0.4 25a 2.6 0.4 36a 2.2 0.1 27a 2.1 0.6 28a 4.6 2.1 29a 1.7 0.4 3

The results represent the cumulative transport of mannitol (% of the initithe apical to the basolateral chamber in the presence of several concentratof 3–22 filters. *, **, *** Results statistically significantly different from buffe

error bars S.D.

effect was also essentially lost at degree of substitutionabove 0.15. Similarly, the N-betainate of glucosamine wasinactive. The onset of the permeation enhancing effect wasrapid, since the mannitol flux was increased in the pres-ence of N-betainate 1a already at 10 min and continuedto rise steadily up to 120 min (Fig. 2). The activity of N-piperazines depended both on the degree of substitution
and the number of the quaternary nitrogens. Again, deriva-tives with lower degrees of substitution were generally moreactive than those with fewer free amine groups. However,the fastest mannitol transport was still considerably lower
e permeation of mannitol

(%) S.D. (%)

2 0.84 0.99 0.61 5.4*

Concentration (w/v)

0.5% 1.0%

n (%) S.D. (%) Mean (%) S.D. (%)

.8 0.6**

.8 0.9

.2 1.0 16.4 4.3***

.6 0.6 7.9 0.9**

.6 0.7 3.2 1.0

.2 0.1 2.8 1.1

.2 1.3 4.4 2.3

.8 1.6 3.4 1.0

.8 0.0 3.5 0.4

.7 0.7 4.0 1.5

.0 0.8 3.1 1.0

al donor) across a confluent Caco-2 cell monolayer in 120 min fromions of chitosan derivatives or controls. The results are mean valuesr control according to Dunn’s test at levels p < 0.05; p < 0.01; p < 0.001.


Table 4 – The effect of N-piperazine derivatives of chitosan on the permeation of mannitol

Mean (%) S.D. (%)

Buffer (pH 7.4) 1.7 0.5

Piperazines Concentration (w/v)

0.25% 0.5% 1.0%

Mean (%) S.D. (%) Mean (%) S.D. (%) Mean (%) S.D. (%)

Chitosan (pH 7.4) 3.7 1.2***1b 2.0 0.3 2.7 1.0 5.0 1.6**2b 1.9 0.7 2.3 0.6 2.5 0.43b 1.5 0.3 1.5 0.5 4.6 0.8**4b 1.9 0.5 2.8 1.1 2.1 0.5

5b 2.6 0.9 3.5 1.3 2.4 0.26b 2.2 0.7 3.1 1.5 3.1 0.77b 2.6 0.8 2.1 0.4 5.5 3.1**8b 2.7 1.0 2.9 0.4 3.1 0.5

9b 4.2 0.9** 6.3 1.7** 9.5 1.3***10b 3.2 0.7 3.9 1.2* 5.3 2.2**11b 4.1 1.6* 3.8 1.3 8.2 3.4***12b 2.3 0.6 3.8 1.8 5.7 2.2**

13b 2.9 1.0 1.8 0.1 2.5 0.814b 2.8 0.9 2.6 1.0 2.2 0.115b 2.8 0.8 2.9 0.8 2.3 0.7

The results represent the cumulative transport of mannitol (% of the initial donor) across a confluent Caco-2 cell monolayer in 120 min fromthe apical to the basolateral chamber in the presence of several concentrations of chitosan derivatives or controls. The results are mean valuesof 5–33 filters. *, **, *** Results statistically significantly different from buffer control according to Dunn’s test at levels p < 0.05; p < 0.01; p < 0.001.

than that achieved with the best N-betainate derivative.It appeared that the diquaternary piperazine derivatives(9b–12b) were more active than the monoquaternary or ter-tiary derivatives. The location of the quaternary nitrogen inthe monoquaternary derivatives (1b–4b and 5b–8b) did notalter the activity to any great extent. Unmodified chitosan(0.5%) could modestly increase mannitol transport at neutralpH (Tables 3 and 4). A lower pH was tested during N-betainateexperiments, and the activity of chitosan increased by 80%when the pH was lowered to 5.5 while buffer control remainedunaffected (Table 3).

3.2. Cytotoxicity

The results of the LDH release test are presented inTables 5 and 6. Fig. 3 shows the correlation between man-nitol transport and LDH release. Unmodified chitosan didnot alter the LDH release. Several N-betainates showed aslight elevation in LDH release that depended on the con-centration and the degree of substitution. The glucosamineN-betainate monomer was non-toxic. LDH release in the pres-ence of monoquaternary and tertiary N-piperazine derivativeswas of similar magnitude as that seen with the N-betainates.Only derivatives 3b, 7b and 13b at the highest concentrationinduced any significant increase in LDH release. The diquater-nary derivatives with low degrees of substitution (9b and 10b)were clearly the most toxic of all these compounds. However,the derivative a with higher molecular weight (11b) evokedLDH release similar to N-betainate 1a. LDH activity in the baso-lateral chamber was also measured after buffer, Triton X-100

Fig. 3 – Correlation between mannitol transport and LDHrelease of chitosan derivatives (1.0% m/v concentration).Solid squares = N-betainates, opendiamonds = monoquaternary N-piperazines with thecharge on the outer nitrogen, opensquares = monoquaternary N-piperazines with the charge
on the inner nitrogen, open triangles = diquaternaryN-piperazines and crosses = tertiary N-piperazines.
and chitosan treatments. The LDH activity was absent fromthe basolateral chambers after buffer or chitosan treatmentsbut Triton X-100 treatment released high levels of LDH activityalso to the basolateral chamber (data not shown).

4. Discussion

There is an intense search for novel, biocompatible perme-ability enhancers to be used in drug delivery. Chitosan is anefficient permeability enhancer but it suffers from poor aque-


Table 5 – The release of lactate dehydrogenase from a confluent Caco-2 cell monolayer during a 120 min permeabilityexperiment in the presence of N-betainate derivatives of chitosan

Controls Mean (%) S.D. (%)

Buffer (pH 7.4) 8.4 3.5EDTA 2.5 mM 6.9 1.1Triton X-100 100.0 41.9***

Betainates Concentration (w/v)

0.25% 0.5% 1.0%


Chitosan 11.3 4.41a 13.4 2.7 22.1 6.2* 30.6 5.5***2a 14.1 3.6 14.8 1.6 23.7 7.5*3a 15.4 5.0 18.1 7.2 23.0 7.4*4a 14.3 3.7 18.3 6.6 27.8 4.7*5a 14.7 2.4 19.0 2.9 28.1 2.9**6a 12.7 7.7 12.1 5.2 15.0 7.77a 15.1 3.5 18.1 1.6 22.1 2.6*8a 11.6 3.3 16.5 6.5 22.0 11.89a 13.8 7.1 10.3 4.9 10.4 3.5

The enzyme activity was measured from the apical chamber and scaled to the maximal value (Triton X-100 treated cells), n = 3–58. *, **, *** Resultsstatistically significantly different from buffer control according to Dunn’s test at levels p < 0.05; p < 0.01; p < 0.001.

ous solubility in the physiological pH range. In this report,two novel families of water-soluble chitosan derivatives, N-betainates and N-piperazines (Tables 1 and 2), were studiedfor their ability to increase paracellular transport across the

Caco-2 cell monolayer. An inverse relationship between thedegree of substitution and the increase in paracellular trans-port was found. N-betainates were found to be less toxic thanN-piperazines.

Table 6 – The release of lactate dehydrogenase from a confluent Caco-2 cell monolayer during a 120 min permeabilityexperiment in the presence of N-piperazine derivatives of chitosan

Controls Mean (%) S.D. (%)

Buffer (pH 7.4) 9.1 7.0Triton X-100 100.0 42.8***

Piperazines Concentration (w/v)

0.25% 0.5% 1.0%


1b 9.4 4.0 16.3 7.2 21.8 1.52b 10.4 5.5 12.6 5.8 14.8 4.83b 7.9 3.7 10.5 2.5 65.2 14.5**4b 10.5 4.6 13.3 5.7 14.6 2.6

5b 13.1 2.5 18.4 1.6 28.2 6.4*6b 23.7 18.0 24.2 11.6 24.1 3.17b 7.1 1.7 10.0 2.4 70.4 12.6**8b 11.7 5.5 14.8 4.7 13.6 1.8

9b 57.3 24.4** 50.1 15.0** 48.4 11.4**10b 31.3 2.5** 42.4 3.3** 45.5 2.7**11b 18.0 3.3 15.0 2.1 26.1 9.312b 17.1 2.4 26.7 1.5* 37.3 3.0**

13b 17.2 3.2 18.0 2.2 34.9 5.2**14b 11.9 4.5 15.8 4.0 25.0 5.315b 25.9 8.5 17.7 5.3 22.2 5.5

The enzyme activity was measured from the apical chamber and scaled to maximal the value (Triton X-100 treated cells), n = 3–58. *, **, *** Resultsstatistically significantly different from buffer control according to Dunn’s test at levels p < 0.05; p < 0.01; p < 0.001.


N-Betainate derivatives showed degree of substitution andconcentration dependent enhancement of mannitol trans-port (Table 3, Fig. 1). Mannitol transport was not significantlyaltered at the two lowest concentrations. However, at the 1.0%concentration mannitol transport increased sharply with N-betainate derivatives 1a (7.5-fold) and 2a (3.5-fold) but notwith molecules with higher degrees of substitution or withthe monomer. Significantly higher increases in mannitol per-meability between chitosan derivatives and buffer controlshave been reported, e.g., for TMC (Thanou et al., 2000; Kotze etal., 1997, 1998) and chitosan (Schipper et al., 1996). However,direct inter-laboratory comparisons on fold-increase valuescannot be made since the apparent permeability of manni-tol in buffer control has been reported in the range of 0.1 to0.5 × 10−6 cm/s (Thanou et al., 2000; Kotze et al., 1997, 1998)while the apparent permeability of mannitol in this study was1.7 × 10−6 cm/s. To illustrate the difference, Kotze et al. (1998)described a 36-fold increase in mannitol permeability in thepresence of chitosan hydrochloride while Triton X-100 (themaximal increase) evoked only a 25-fold increase in manni-tol transport in the present study. The onset of action of thestudied derivatives is rapid (Fig. 2), as has also been reportedfor TMC (Kotze et al., 1997) and chitosan (Schipper et al., 1996).

An increase in the degree of substitution decreased theamount of regular chitosan subunits. This increases the watersolubility but, at the same time, putatively bioactive partsof the molecule become sterically protected. This is consis-tent with published studies on deacetylated chitosan sincethe bioactivity tends to increase as the degree number ofacetylated amine groups decreases (Schipper et al., 1996). Forunmodified chitosan, a high positive charge of chitosan back-bone is essential to achieve activity (Artursson et al., 1994).Thus, it is not the number of positive charges per se but thecombination of amount and location of charge is probably cru-cial for the bioactivity of chitosan because the highly chargedN-betainates are virtually ineffective. On the other hand, thebiological activity of TMC has been shown to increase as thedegree of substitution increases (Thanou et al., 2000; Kotze etal., 1999a,b). However, the structure of TMC is much closer tothe structure of intact chitosan than the N-betainate deriva-tive and the cationic group of TMC may act similarly to chargedamino group of unmodified chitosan.

A concentration of 1.0% of N-betainate derivatives 1a and2a was needed before it was possible to observe any clear bio-logical effect. This is higher than effective concentrations ofTMC at pH 6.2 and 7.2 (Kotze et al., 1998; Thanou et al., 2000).Regular deacetylated chitosans also show effect on monolayerintegrity even at 0.05% concentration but a low pH value isneeded (Schipper et al., 1996).

It has been previously shown that the absorption enhance-ment effect of chitosan does not increase with increasingconcentrations in the neutral pH (Kotze et al., 1999a,b). This isdue to insolubility of unmodified chitosan in the neutral con-ditions. Thus, we concluded that one chitosan concentration,i.e., 0.5%, was sufficient for control. In the present study, 0.5%chitosan was only modestly effective at pH 7.4 in which it isonly weakly charged and poorly soluble, but the activity nearlydoubled as the pH was lowered (Table 3).

All N-betainate derivatives displayed a modest and concen-tration dependent increase in LDH release indicating minor

cytotoxicity. This kind of behavior is also typical of deacety-lated chitosans (Schipper et al., 1996; Dale et al., 2006) andchitosan glutamate (Chae et al., 2005). The absolute amountsof released LDH at equal concentrations were modestly higherwith N-betainates than with regular chitosan. This is prob-ably explained by differences in the dissolved concentrationsince chitosan is very poorly whereas N-betainates are verywell soluble at pH 7.4. Previous studies using Caco-2 cellsbut different protocols have reported LDH release values ofup to 40% in 3 h for 0.5% chitosan (Silva et al., 2006) and35% release in 2 h for 0.1% TMC (Mao et al., 2005). Anotherstudy also reported that 0.5% chitosan caused a 3-fold increasein LDH release over buffer control within 30 min. Nonethe-less, the trypan blue exclusion test indicated no toxicity,suggesting that the cells had not been seriously damaged(Dodane et al., 1999). The absolute LDH release percentagesmay seem relatively high but it should be borne in mind thatthe experiments were performed with well differentiated cellsgrown on polycarbonate filters and not on relatively undif-ferentiated cells grown on plastic in multi-well plates. Thepositive control (Triton X-100) lyses the whole cell popula-tion, and the released LDH can distribute freely into bothchambers which dilutes this positive control as it was sam-pled from the apical chamber. In contrast, the monolayerretains its structure rather well in the presence of chitosanderivatives and the released LDH is mainly restricted to theapical chamber. Taken together, the absolute level of LDHrelease induced by N-betainates does not point to any serioustoxicity.

In addition to differences in molecular weight and thedegree of substitution, N-piperazine derivatives possessed dif-ferent numbers (0, 1 or 2) of positive charges per monomer(Table 2). The number of cationic charges affected both thetransport enhancing activity (Table 4, Fig. 1) and LDH release(Table 6) of these derivatives. An increase in the numberof charges per monomer resulted in both higher activityand LDH release. It appeared that piperazine derivativesinduced more extensive LDH release than betainates whenthe desired bioactivity increased (Fig. 3). Diquaternary deriva-tives (9b–12b) displayed the strongest whereas the tertiaryderivatives (13b–15b) exhibited the weakest activity on para-cellular transport. No clear difference was seen between themonoquaternary derivatives with different locations of thecharge (1b–4b and 5b–8b). The high activity attained withthe N-betainate derivative 1a was not achieved with anyN-piperazine derivative. However, N-piperazine derivativeswith such low degrees of substitution were not synthesized.When derivatives with similar substitution levels are com-pared (Fig. 1), equal or even higher activities are observed withthe N-piperazines. These results also support the importanceof the unmodified chitosan backbone for bioactivity when rel-atively large substituents are used.

LDH release after treatment with N-piperazines was gener-ally similar to treatments with N-betainates (Tables 5 and 6).Only the two diquaternary derivatives with the lowest degreeof substitution (9b and 10b) seemed somewhat toxic at allconcentrations. The sharp increase in toxicity of 3b and7b at concentrations above 0.5% is still unexplained. Thetoxicities of N-piperazines are generally acceptable, at leastin vitro.


5. Conclusions

The quaternary chitosan derivatives studied show good aque-ous solubility at neutral pH. The aqueous solubility of thederivatives enable more formulation possibilities comparedto poorly soluble unmodified chitosan. When the degree ofsubstitution is kept low at physiological pH, these novelderivatives possess the same bioactivity that chitosan dis-plays at acidic pH. The toxicity of both groups of compoundswas generally modest. Overall, the inverse proportionalitybetween the degree of substitution and activity suggests thatan intact chitosan backbone is essential for the bioactivity ofchitosan derivatives. The permanent positive charge of qua-ternary betaine or piperazine group does not substitute forthe charge at the free amine group. This was especially clearwith the betainate derivative, i.e., it can be concluded that chi-tosan N-betainates should contain only the minimum numberof substituents required to guarantee water solubility in orderto reach the highest activity.

Acknowledgement

This study was financially supported by Finnish FundingAgency for Technology and Innovation (TEKES).

r e f e r e n c e s

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