Enhancement of paracellular drug transport with highly quaternized N-trimethyl chitosan chloride in neutral environments: In vitro evaluation in intestinal epithelial cells (Caco-2)

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  • Enhancement of Paracellular Drug Transport with Highly QuaternizedN-Trimethyl Chitosan Chloride in Neutral Environments: In VitroEvaluation in Intestinal Epithelial Cells (Caco-2)


    Contribution from Department of Pharmaceutics, Potchefstroom University for Christian Higher Education,Potchefstroom, 2520, Republic of South Africa, Department of Pharmaceutical Technology, Leiden/Amsterdam Center for DrugResearch, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, LTS Lohmann Therapie-Systeme GmbH,D-56605 Andernach, Germany, and Department of Pharmacology, Leiden/Amsterdam Center for Drug Research, LeidenUniversity, P.O. Box 9503, 2300 RA Leiden, The Netherlands.

    Received June 1, 1998. Accepted for publication October 2, 1998.

    Abstract 0 Previous studies have established that a partiallyquaternized derivative of chitosan, N-trimethyl chitosan chloride (TMC),can be used as an absorption enhancer for large hydrophiliccompounds across mucosal surfaces. This study evaluates andcompares the effects of the degree of quaternization of TMC, in aneutral environment, on the permeability of intestinal epithelial cellsin vitro, where normal chitosan salts are ineffective as absorptionenhancers. The effects of TMC-H [61.2% quaternized, (0.051.5%w/v)], TMC-L [12.3% quaternized, (0.51.5% w/v)], and chitosanhydrochloride [0.51.5% w/v] on the transepithelial electrical resistance(TEER) and permeability, for the hydrophilic model compound [14C]-mannitol, of intestinal epithelial Caco-2 cell monolayers, wereinvestigated at pH values of 6.20 and 7.40. The viability of themonolayers was checked with the trypan blue exclusion technique.At a pH of 6.20, all the polymers caused a pronounced reduction(3767% at 0.5% w/v concentrations) in the TEER of Caco-2 cells.On the contrary, at a pH of 7.40, only TMC-H was able to decreasethe TEER values, even in a concentration as low as 0.05% w/v (35%reduction). Comparable results were obtained with the permeation of[14C]mannitol. Large increases in the transport rate (1823-fold at0.5% w/v concentrations) were found at pH 6.20, whereas only TMC-Hwas able to increase the permeation of [14C]mannitol at pH 7.40 (3148-fold at 0.051.5% w/v concentrations of TMC-H). For all thepolymers studied, no deleterious effects to the cells could bedemonstrated with the trypan blue exclusion technique. It is concludedthat highly quaternized TMC is a potent absorption enhancer and thepotential use of this polymer, especially in neutral and basicenvironments where normal chitosan salts are not effective, is expectedto be an important contribution to the development of effective deliverysystems for hydrophilic compounds such as peptide drugs.


    The potential use of chitosan as an absorption enhanceracross mucosal surfaces has been well documented inrecent years. Chitosan salts such as chitosan glutamate

    and chitosan hydrochloride have been shown to increasethe absorption of a number of hydrophilic compounds andpeptide drugs both in vitro and in vivo.1-7 Apart from itsmucoadhesive properties,8 chitosan acts mainly by openingthe tight junctions between epithelial cells to allow for theparacellular transport of these large hydrophilic molecules.Such an action is believed to be due to an interaction of apositively charged amino group on the C-2 position ofchitosan with negatively charged sites on the cell mem-branes, which results in a structural reorganization of thetight junction-associated proteins.1,6

    Chitosan has an apparent pKa value between 5.5 and6.5, and a certain amount of acid is required to transformthe glucosamine units into the positively charged, water-soluble form. At neutral and basic pH values, the chitosanmolecules will lose their charge and therefore the potentialuse of this polymer, especially in more neutral and basicenvironments such as those found in the large intestineand colon, is limited. It has been shown recently that apartially quaternized (12%) derivative of chitosan, N-trimethyl chitosan chloride (TMC), was also able to sig-nificantly increase the transport of hydrophilic compounds,such as [14C]mannitol (MW 182.2), fluorescein isothio-cyanate-labeled dextran (MW 4400), and [14C]poly(ethyleneglycol) (MW 4000), and the peptide drugs buserelin (MW1300), 9-desglycinamide, 8-L-arginine vasopressin (MW1412), and insulin (MW 5778) in Caco-2 cell monolayersat acidic pH values (4.40-6.20).9-11 It was suggested thatTMC most likely has the same mechanism of action on thejunctional complex as other chitosan salts.10 The derivativeTMC (12% quaternized) was not as effective as otherchitosan salts, such as chitosan glutamate and chitosanhydrochloride. This lesser efficacy was explained by itscharge density, which was determined by the degree ofquaternization, and by a partial hiding of the positivecharge on the amino group by the attached methylgroups.10,11 However, the much higher aqueous solubilityof TMC may compensate for its lesser efficacy. TMC hasproved to be very soluble over a wide pH range (pH 1-9)up to 10% w/v concentrations, even at degrees of quater-nization as low as 10%.9-10

    Our hypothesis is that TMC with higher degrees ofquaternization may be more effective as an absorptionenhancer to increase the paracellular transport of hydro-philic compounds at neutral pH values. The aim of thepresent study was to synthesize TMC with differentdegrees of quaternization (low and high) and to evaluateand compare the effect of these polymers with the effect ofchitosan hydrochloride on the permeability of intestinal

    * Corresponding author. Telephone: 27 18 2992249. Fax.: 27 182992251. E-mail: FMSAFK@PUKNET.PUK.AC.ZA.

    Department of Pharmaceutics, Potchefstroom University for Chris-tian Higher Education.

    Department of Pharmaceutical Technology, Leiden/AmsterdamCenter for Drug Research.

    LTS Lohmann Therapie-Systeme GmbH. Department of Pharmacology, Leiden/Amsterdam Center for Drug


    1999, American Chemical Society and 10.1021/js980233c CCC: $18.00 Journal of Pharmaceutical Sciences / 253American Pharmaceutical Association Vol. 88, No. 2, February 1999Published on Web 12/05/1998

  • epithelial cells in vitro for increased transport of a hydro-philic compound at slightly acidic and neutral pH values(6.20 and 7.40).

    Experimental SectionSynthesis and Characterization of N-Trimethyl Chitosan

    Chloride (TMC)sTwo batches of TMC, one with a low and onewith a high degree of quaternization, were synthesized from sievedfractions (

  • Effect on the TEER of Intestinal Epithelial CellssThe effects of 0.5% w/v concentrations of TMC-H, TMC-L,and chitosan hydrochloride on the TEER of the Caco-2 cellmonolayers at pH 6.20 and 7.40 are shown in Figures 1Aand 1B, respectively. At pH 6.20, incubation with thesepolymers resulted in a pronounced reduction in TEERvalues compared with the control group. After 2 h incuba-tion, the reduction in TEER was in the following order:chitosan hydrochloride (67.1 ( 0.6%) > TMC-H (55.9 (3.1%) > TMC-L (37.3 ( 1.4%). Higher polymer concentra-tions (1.0 and 1.5% w/v) did not cause any further signifi-cant decreases in the TEER.

    At pH 7.40, only TMC-H was able to decrease the TEERof the cell monolayers (Figure 1B). Neither TMC-L norchitosan hydrochloride, even in concentrations of 1.5% w/v,was able to cause any significant decreases in TEER valuescompared with the control group. Incubation of the mono-layers with TMC-H resulted in a pronounced decrease inthe TEER of the cell monolayers. After 2 h incubation, thereduction in TEER was as followed: 43.1 ( 2.5% at 0.1%w/v TMC-H, 45.6 ( 3.7% at 0.25% w/v TMC-H, 58.6 ( 0.7%at 0.5% w/v TMC-H, 60.1 ( 3.1% at 1% w/v TMC-H, and63.1 ( 2.1% at 1.5% w/v TMC-H. Even in a concentrationas low as 0.05% w/v, TMC-H was able to reduce the TEERby 34.8 ( 4.1%.

    Reversibility of the effect of 0.5% w/v concentrations ofthese polymers on TEER could not be demonstrated at anypH value (Figures 1A and 1B). Complete removal of thepolymers, without damaging the cells, proved to be prob-lematic due to the high viscosity and mucoadhesivity of thepolymer solutions. Furthermore, the repeated washing of

    the cells and the short time allowed for recovery may alsobe reasons why significant reversibility of the effect couldnot be demonstrated. However, in concentrations

  • of the cells was found when cells were stained with thisdye after completion of all the transport studies.

    DiscussionThe results of this study show that both chitosan

    hydrochloride and TMC, with different degrees of quater-nization, are potent absorption enhancers at a pH of 6.20.These polymers were able to decrease the TEER of theCaco-2 cell monolayers markedly. Measurement of TEERis believed to be a good indication of the tightness of thejunctions between epithelial cells. The apparent differencein effect between chitosan hydrochloride and TMC couldbe explained in terms of their respective charge densities.The amino group on the C-2 position of TMC is a muchlarger unit, due to the attached methyl groups, comparedwith the amino group on the C-2 position of chitosanhydrochloride. With chitosan hydrochloride, about 10 wt% constitute the salt part and therefore the charge densityis much higher as for TMC. Additionally, the attachedmethyl groups on TMC may partially shield the positivecharge and steric effects may also influence the configu-ration of the TMC molecule where chitosan hydrochlorideprobably exists in a linear configuration at acidic pH valueswhen the amino group is protonated. A clear comparisonbetween these polymers is not possible because the equiva-lent amounts of free chitosan base could not be calculated,especially for TMC because NMR spectra showed that someamino groups are still dimethylated, depending on thedegree of quaternization of the respective TMC sample.

    In agreement with the reduction in TEER at a pH of6.20, the permeation of the hydrophilic model compound[14C]mannitol was also increased markedly, indicating

    enhanced paracellular transport. Because of their positivecharge, cationic macromolecules such as chitosan and TMCcan interact with the anionic components of the glycopro-teins on the surface of epithelial cells. Cationic macro-molecules can displace cations from electronegative siteson cell membranes, which require coordination with cationsfor dimensional stability.16 It is also known that the interiorof the tight junction channel are hydrated and containedfixed negative sites. Changes in the concentration of certainions in the pore could result in alteration in tight junctionresistance leading to opening of the pore with increasedparacellular permeability.17 It has been reported thatchitosan is able to induce a redistribution in cytoskeletalF-action, thereby resulting in a structural reorganizationof tight junction associated proteins such as ZO-1.1,6 TMCprobably acts in a similar way to open the tight junctions.10

    At a pH of 7.40 only TMC-H was able to decrease theTEER of the cell monolayers. In agreement with thisreduction in measured TEER, the permeation of [14C]-mannitol was substantially increased even in concentra-tions as low as 0.05% w/v. Chitosan hydrochloride has anapparent pKa value of 5.5-6.5 and, at a pH of 7.40, itprobably exists in a more coiled configuration with noprotonated amino groups that can interact with the cellsurfaces or tight junctions. With TMC-L, the chargeddensity has not reach the threshold concentration to induceinteraction with the anionic components of the glycopro-teins at the surface of the cells or with the fixed negativecharges within the aqueous tight junctions. Additionally,the attached methyl groups may partially shield thepositive charge from significant interaction with the cellmembranes or tight junctions. TMC-H, on the other hand,has a much higher proportion of quaternary amino groupsthat seems to be sufficient to interact with anionic com-ponents on the cell membranes or the negative sites withinthe tight junctions.

    Because of the high viscosity and mucoadhesive char-acter of chitosan hydrochloride and TMC, it is unlikely thatall the polymer solution could be removed without damag-ing the cells. Therefore, reversibility of the effect of thesepolymers on the TEER could not be established. Neverthe-less, the absence of intracellular trypan blue staining, afterprolonged incubation with these polymers, implies that theCaco-2 cells remained undamaged and functionally intact.This implication is in agreement with results obtained ina recent study where Caco-2 cell monolayers were testedwith the propidium iodide nucleic stain for their viabilityafter 4 h of incubation with TMC (40 and 60% quaternized)at different concentrations up to 1.0% w/v. In all cases, themonolayers were able to exlude propidium iodide.18

    Table 1sEffect of TMC-H, TMC-L, and Chitosan Hydrochloride on the Permeability of [14C]Mannitol

    TMC-H TMC-L chitosan hydrochloride

    pH polymer concentration (% w/v) Papp 10-7 (cm s-1)a R Papp 10-7 (cm s-1) R Papp 10-7 (cm s-1) R

    6.20 control 0.72 0.09 1 0.72 0.09 1 0.72 0.09 10.10 12.81 2.56b 18 n.d.c n.d.0.25 13.32 0.58b 19 n.d. n.d.0.50 14.28 2.14b 20 13.15 1.54b 18 16.38 0.68b,d 231.00 16.14 0.72b 22 10.73 1.87b 15 20.82 1.07b,d 291.50 14.44 1.80b 20 11.32 0.80b 16 31.03 3.16b,d 43

    7.40 control 0.47 0.04 1 0.47 0.04 1 0.47 0.04 10.05 14.69 2.90b 31 n.d. n.d.0.10 14.06 2.85b 30 n.d. n.d.0.25 15.10 2.20b 32 n.d. n.d.0.50 20.14 1.44b 43 0.63 0.08 1 0.56 0.09 11.00 22.57 1.89b 48 0.85 0.08 2 0.51 0.02 11.50 15.13 1.76b 32 1.07 0.89 2 0.67 0.04 1

    a Papp ) apparent permeability coeficient; each value represents the mean SD of 3 experiments. b Significantly different from control (p < 0.05). c n.d., Notdetermined. d Significantly different from all other treatmeants in group (p < 0.05).

    Figure 3sThe effect of different concentrations of TMC-H on the cumulativetransport of [14C]mannitol in Caco-2 cell monolayers at a pH of 7.40. Eachpoint represents the mean SD of three experiments.

    256 / Journal of Pharmaceutical SciencesVol. 88, No. 2, February 1999

  • In summary, our study shows that the insolubility ofchitosan and chitosan salts prevents these polymers frombeing effective as absorption enhancers at neutral pHvalues. It has been shown that TMC, already at very lowdegrees of quaternization, is superior in water solubilitycompared with chitosan and chitosan salts.9,10 The degreeof quaternization of TMC is demonstrated to play animportant role in its ability to open the tight junctions ofintestinal epithelial cells. Highly quaternized TMC provesto be a very potent absorption enhancer, especially atneutral pH values. The use of this chitosan derivative maybe a valuable contribution to the development of selectiveand effective delivery systems for hydrophilic compoundssuch as peptide drugs, especially in neutral and basicenvironments where normal chitosan salts are ineffective.

    References and No...


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