effect of degree of quaternization of n-trimethyl chitosan chloride for enhanced transport of...

Journal of Controlled Release 64 (2000) 15–25www.elsevier.com/ locate / jconrel

Effect of degree of quaternization of N-trimethyl chitosanchloride for enhanced transport of hydrophilic compounds

across intestinal Caco-2 cell monolayersa a,b a c d´M.M. Thanou , A.F. Kotze , T. Scharringhausen , H.L. Lueßen , A.G. de Boer ,

a a ,*J.C. Verhoef , H.E. JungingeraDepartment of Pharmaceutical Technology, Leiden /Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502,

2300 RA Leiden, The NetherlandsbDepartment of Pharmaceutics, Potchefstroom University for Christian Higher Education, Potchefstroom, 2520,

Republic of South AfricacOctoPlus, Leiden, The Netherlands

dDepartment of Pharmacology, Leiden /Amsterdam Center for Drug Research, Leiden University, P.O. Box 9503, 2300 RA Leiden,The Netherlands

Received 1 April 1998; accepted 15 April 1999

Abstract

N-Trimethyl chitosan chloride (TMC) is a permanently quaternized chitosan derivative with improved aqueous solubilitycompared to native chitosan. TMC is able to open the tight junctions of intestinal epithelia at physiological pH values, wherechitosan is insoluble and therefore ineffective. TMCs with degrees of substitution of 40 and 60% were synthesized accordingto a novel synthesis procedure and their effect on the permeability of the tight junctions of the intestinal Caco-2 monolayerswas studied, measuring the transepithelial electrical resistance and the transport of a mainly paracellularly transported

14compound, [ C]-mannitol. Toxicity studies using nucleic stains were done to establish the transport as a cause of opening ofthe tight junctions and not of possible cytotoxicity. TMC60 showed higher transport enhancement ratios than TMC40 in allconcentrations tested (0.05–1.0%, w/v). Both derivatives did not affect the viability of the Caco-2 cell monolayers. Theseresults suggest that high charge density is necessary for TMC to substantially improve the paracellular permeability ofintestinal epithelia. It is expected that TMC40 and TMC60 will enhance the intestinal permeation of hydrophilicmacromolecular drugs such as peptides and proteins. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Caco-2 monolayers; Trimethyl chitosan chloride; Degree of substitution; Tight junctions; Absorption enhancers

1. Introduction high interest as a research object. Low oral bioavail-ability of peptide and protein drugs can be accepted

Oral peptide and protein drug delivery attracts as far as its reproducibility is guaranteed. Targetingof these agents to gastrointestinal sites where lumi-nal, brush border and intracellular proteolytic ac-*Corresponding author. Tel.: 131-71-527-4308; fax: 131-71-

527-4565. tivities are low (for example, colon) can be a

0168-3659/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 99 )00131-5

16 M.M. Thanou et al. / Journal of Controlled Release 64 (2000) 15 –25

solution to the degradative enzyme barrier [1]. The amine units ((1,4)-linked poly(2-amino-2 deoxy-b-D-use of peptidomimetics that show higher stability glucose or poly (D-glucosamine)) and has an appar-towards the degradative enzymes can be another ent pK of 5.5, as measured by potentiometrica

approach to overcome this barrier. Combinatorial titration. The polymer is therefore only soluble insynthesis of large libraries of peptides is a powerful acidic solutions (pH 1–6) where most of the aminotool for the design of non-peptide drugs. However, groups are protonated. Recent studies have showneven if these hydrophilic agents manage to overcome that only protonated soluble chitosan i.e. in itsthe luminal and cytosolic degradation, still the uncoiled configuration can trigger the opening of thebarrier of the epithelial tight junctions of the intestine tight junctions, thereby facilitating the paracellularremains of great importance for the regulation of the transport of hydrophilic compounds [23]. This prop-paracellular absorption of these high molecular erty implies that chitosan would be effective as anweight hydrophilic compounds. absorption enhancer only in a limited area of the

To overcome this epithelial transport barrier, intestinal lumen where the pH values are close to itsseveral classes of absorption enhancers have been pK (e.g. proximal duodenum). For that reason, ita

evaluated [2–5] such as fatty acids, bile salts, ionic may not be a suitable carrier for targeted peptideand non-ionic surfactants, acyl carnitines and drug delivery to specific sites of the intestine.cholines, salicylates, chelating agents and swellable To overcome this problem a chitosan derivativepolymers like starch, polycarbophil, and chitosan. In N,N,N-trimethyl chitosan chloride (TMC) has beensome of these cases cell toxicity and enhancing synthesized and characterized [24,26]. TMC showseffect has been found to be strongly related [6,7]. For higher solubility than chitosan in a broader pH range.this reason polymers of natural origin have attracted TMC can be quaternized in different degrees,a lot of interest in research for their use as peptide dependent upon the conditions, steps and duration ofdrug absorption enhancers. One of these polymers is the synthesis reaction [24]. The degree of molarchitosan which is the second most abundant natural substitution is expected to play an important role onoccurring organic material after cellulose and can be the properties of TMC to increase the permeability ofobtained from chitin, a waste material from the sea the intestinal epithelia. Special care has to be takenfood industry, by alkaline or enzymatic deacetyla- to study the toxicity of these quaternized chitosanstion. Chitosan’s biocompatibility and biodegradation and to differentiate the enhancing effect from pos-[8,9] suggest its safe use. Chronic nasal application sible toxicity properties. The aim of the present studyof chitosan in guinea pigs has been studied, showing is to synthesize N-trimethyl chitosan chloride with anno substantial effect on the ciliary beat frequency intermediate degree of substitution and to compare it[10]. Chitosan has been investigated in the pharma- with the high degree of substitution derivative con-ceutical field as the main excipient in granules and cerning its effect on the permeability and integrity oftablets [11,12], gels, coacervates or microspheres intestinal Caco-2 cell monolayers.[13–16] and recently sponge-like formulations ofchitosan have been referred to as sustained releasedrug carriers [17]. In repeated adhesion studies, it 2. Materials and methodshas been proven by Lehr et al. [18] that chitosan isfairly mucoadhesive in comparison to polycarbophil. 2.1. MaterialsChitosan has also been demonstrated to promote thenasal absorption of insulin in rats and sheep [19,20] Chitosan (Seacure 210, 93% deacetylated) was aand to enhance the paracellular transport of peptides gift from Pronova Biopolymer A.S. (Drammen,in vitro and in vivo by opening of the tight junctions Norway). N-Methylpyrrolidinone was obtained from

14[21,22]. Acros (Geel, Belgium). [ C]-Mannitol (M 182.2;w

Despite all these properties, chitosan is still a specific radioactivity 57 mCi /mmol) was obtainedpolymer that lacks the advantage of good solubility from Amersham Life Science (Little Chalford, UK).at physiological pH values. It aggregates in solutions Sodium dodecyl sulfate (SDS) was obtained fromat pH values above 6. Chitosan consists of glucos- Baker (Deventer, The Netherlands), trypan blue from

M.M. Thanou et al. / Journal of Controlled Release 64 (2000) 15 –25 17

Sigma (Bornem, Belgium), and propidium iodidefrom Molecular Probes (Leiden, The Netherlands).

2.2. Synthesis and characterization of N-trimethylchitosan (TMC) with different degrees ofsubstitution

TMCs with degrees of substitution of 39%(TMC40) and 65% (TMC60) were synthesized aspreviously described [24]. Briefly, sieved chitosan(particle size 200–400 mm) was mixed with methyl-iodide in a basic solution of N-methylpyrrolidinoneat 608C for 75 min. The product was isolated byethanol precipitation and subsequent centrifugation.After this first step the obtained product underwent asecond step of reductive methylation, to yield thefinal products TMC iodide having 39% degree ofsubstitution after 60 min and TMC iodide with adegree of substitution 65% after 90 min. Bothproducts were precipitated by addition of ethanol andisolated by centrifugation. The purification step ofthe final products included the exchange of thecounterion iodide with chloride. The products weredissolved in NaCl containing aqueous solutions, re-precipitated by ethanol, isolated by centrifugationand thoroughly washed with ethanol and diethyl-ether.

Both TMC40 and TMC60 were characterized by1H-NMR (Fig. 1a and b). The products were dried invacuo and measured in D O at 808C, using a 300- or2

a 600-MHz spectrometer (Bruker, Switzerland).

1Fig. 1. H-NMR spectra of (a) TMC 40 and (b) TMC60. Peak2.3. Cell cultures 1assignment: –N (CH ) : 3.4 ppm; –N(CH ) : 2.6 ppm.3 3 3 2

Caco-2 cell cultures of passage numbers 73–84were used for all of the experiments. The cells were medium was changed every second day. The cellseeded on tissue cultured polycarbonate membrane cultures were kept at a temperature of 378C, in an

2 2filters (pore size 0.4 mm, area 4.7 cm and 0.33 cm ) atmosphere of 10% CO and 95% humidity. For all2

in Costar Transwell 6- and 24-well plates (Costar the experiments cells were used 23–25 days afterEurope, Badhoevedorp, The Netherlands) at a seed- seeding. Two hours before the experiments the

4 2ing density of 10 cells /cm [4]. Dulbecco’s Modi- medium was changed to DMEM buffered to pH 7.4fied Eagle’s Medium (DMEM, Sigma, pH57.4), with 25 mM n-(2-hydroxyethyl) piperazine-N-(2-supplemented with 1% non-essential amino acids, ethanosulfonic acid) (HEPES).10% fetal calf serum (Hyclone, Gibco, The Nether-lands), benzyl-penicillin G (160 U/ml) and strep- 2.4. Measurements of transepithelial electricaltomycin sulfate (100 mg/ml) (both obtained from resistance (TEER)Sigma) was used as culture medium, and added toboth the donor and the acceptor compartment. The A Millicell ERS meter (Millipore Corp., Bed-


ford, MA, USA) connected to a pair of chopstick P 5 (dc /dt) ? (1 /A.60.C )app o

electrodes was used to measure the TEER. The effectwhere P is the apparent permeability coefficientof the TMCs on the monolayer was monitored every app

(cm/s), dc /dt the permeability rate, A the diffusion30 min. Preparations of the polymers in DMEM2area of the monolayer (cm ) and C the initialbuffered with HEPES were adjusted at both pH 6.2 o

concentration of the radiomarker. Transport enhance-and 7.2 with aqueous solutions of 0.1 N HCl and 0.1ment ratios R were calculated from P values:N NaOH, respectively. One hour prior to the apical app

application of the polymers, the resistance of the R 5 P /Papp polymer app controlmonolayers was recorded. After 2 h of applicationthe polymers were removed from the apical side and

2.6. Trypan blue exclusionthe cells were washed gently to avoid detachment ofthe cells from the polycarbonate filter. The resistance 14After the TEER and [ C]-mannitol transportwas recorded for one additional hour to study the

experiments, both the apical and basolateral side ofreversibility. Experiments were done in triplicate atthe monolayers were rinsed gently with sterile 0.01378C in an atmosphere of 95% humidity and 10%M phosphate buffered saline (PBS, pH 7.4). At theCO .2 apical side the buffer was substituted with 0.1%

14 solution of trypan blue in PBS, and the monolayers2.5. Permeability studies with [ C]-mannitolwere incubated for 30 min. The solutions wereremoved from both sides, and the monolayers wereThe permeability of the monolayers under therinsed gently to remove the excess of the dye. Theinfluence of the polymers was tested by measuring

14 support filters with the cells were cut carefully fromthe transport of the radiomarker [ C]-mannitol. Two14 the inserts and transferred on coverslips. The cells onhours before the application of the [ C]-mannitol

the filters were checked by light microscopy forpreparations, the Caco-2 cells were transferred toexclusion of the trypan blue and the percentage ofDMEM-HEPES without serum at pH 7.4. Thestained cells was estimated. Cells that excludedmonolayers were incubated at the apical side with a

14 trypan blue were considered to be viable.solution of [ C]-mannitol (4 mmol / l, specific activi-ty 0.2 mCi/ml) in DMEM without serum bufferedwith HEPES at a concentration of 40 mmol / l. The 2.7. Viability of the cells using propidium iodidepolymers TMC40 and TMC60 were dissolved in theabove mentioned solution at concentrations ranging Caco-2 cells were incubated for 4 h at the apicalfrom 0.05 to 1.0% (w/v) and the pH was adjusted at side with and without (control) the polymers TMC606.2 or 7.2. Samples of 200 ml were taken for 4 h and TMC40 at concentrations of 1.0% in serum freefrom the basolateral side. The sample volumes were DMEM buffered with HEPES. Thereafter the poly-replaced with equal volumes of DMEM-HEPES pH mers were removed, the monolayers were rinsed7.4. All experiments were done in triplicate in an apically and basolaterally with sterile PBS, and aatmosphere of 95% humidity and 10% CO at 378C. solution of propidium iodide at a concentration of 302

Radioactivity in the samples was determined after mg/ml in DMEM-HEPES was applied apically for 3addition of 3 ml scintillation cocktail (Ultima-Gold, min. The apical and the basolateral solutions werePackard Instruments, Groningen, The Netherlands), removed, and the support filter with the monolayerusing a liquid scintillation counter (Tri-Carb 1500, was carefully cut from the plastic insert and trans-Packard). Amounts of 200 ml of DMEM-HEPES ferred on two glass round coverslips. The monolayerwithout the radioactive marker were used to de- was sandwiched between the coverslips and thentermine the background radioactivity. Results were mounted on a heating microscope stage (378C) of acorrected for the dilution and expressed as cumula- confocal laser scanning microscope [5].tive transport with time. Apparent permeability co- An MRC-600 Lasersharp system (Bio-Rad Lab-efficients were calculating according to the following oratories, Richmond, CA, USA) linked to a Zeiss IMequation: 35 inverted microscope (Carl Zeiss, Oberkochen,


Germany) was used to visualize the monolayer. 3.2. Effects on TEERPropidium iodide was excited at 514 nm. Cellswhich excluded propidium iodide were considered to The results of the TEER measurements of 0.25%be viable. The experiments were done in triplicate. (w/v) TMC40 and TMC60 for pH values of 6.2 andAn image of damaged cells was used for comparison 7.2 are depicted in Fig. 2. Table 1 gives the decreaseof the viability of the cells. In that case the cells of the initial TEER values at different concentrationswere incubated with 0.1% SDS for 10 min and 30 after 120 min incubation of the monolayers withmg/ml propidium iodide for the last 3 min. these polymers. Both TMC40 and TMC60 were able

to significantly decrease the resistance of the mono-layers at all concentrations (0.05–1.0%) and pHvalues measured. From Table 1 it is evident that the

3. Results effect of the polymers on the TEER of the mono-layers is concentration-dependent. TMC40 managedto decrease the TEER in a more pronounced way

3.1. Synthesis and characterization of TMCsthan TMC60. No major differences on the decreaseof TEER were observed for the two pH values as1In Fig. 1 typical H-NMR spectra of TMC40 anddepicted in Table 1. From Fig. 2 it is also clear that

TMC60 are depicted. The calculation of the degreethe resistance values show a tendency to recover1of substitution was performed by using the H-NMRwhen after 2 h application the medium containing

spectra assigning the quaternized peak at 3.4 ppmTMC was removed. Complete recovery was not

and the tertiary peak at 2.6 ppm [24]. The integral ofobserved, most likely due to the mucoadhesiveness

the quaternized peak for TMC40 (Fig. 1a) andof these polymers. At the end of the experiments all

TMC60 (Fig. 1b) was 7.46 and 10.47, respectively.monolayers excluded the trypan blue staining, in-

The degree of quaternization was calculated accord-dicative of the retained viability of the cells.

ing to the following formula:

143.3. Effects on [ C]-mannitol transportD.S. 5E / ESpeak at 3.4 ppm peak at 4.7 ppm

14The cumulative transport of the radiomarker [ C]-1E 3 9.D mannitol under the influence of 0.25% TMC40 andpeak at 5.4 ppm

TMC60 at both pH 6.2 and 7.2 is presented in Fig. 3.The peaks at 4.7 and 5.4 ppm are assigned as H-1 After 4 h of incubation with TMC40 and TMC60 the

proton of chitosan and are used as internal standards transport was increased substantially at both pH[25]. values. At the concentration of 0.25%, TMC60 was

The duration of the second reaction step was about twice as potent as TMC40. From the mannitolcritical for the obtained degree of the quaternization transport data the apparent permeability coefficientsof chitosan. Higher reaction times or repeating the were calculated (Table 2) and enhancement ratiosreaction a third time yielded products with a degree were subsequently plotted for the different concen-of substitution above 80%. These products, however, trations of TMC40 and TMC60, as depicted in Fig. 4showed less aqueous solubility because of the ex- for both pH values. It is evident that both TMCstended additional methylation of the hydroxyl groups display higher enhancing effect at a more neutral pHat the 3 and 6 position. The TMC solutions were less value. At pH 7.2 TMC40 elicits the highest enhance-viscous than the acidic (pH 4) chitosan solution at ment ratio, (20.0) at 1.0% concentrations, while forthe same concentrations (data not shown). The TMC60 at the same concentration of 1.0% themeasured aqueous solubility of the quaternized de- enhancement ratio is 34.6. At the end of the transportrivatives TMC40 and TMC60 up to concentrations of experiments all monolayers excluded the nucleic dye10% (w/w) was not dependent on the pH of the trypan blue, demonstrating the viability of the cellssolutions. during the transport of mannitol.


Fig. 2. Effect of 0.25% (w/v) TMC 40 and TMC60 on TEER of Caco-2 cell monolayers at pH values of 6.2 and 7.2. At 120 min the cellswere washed and replaced in pure culture medium. (s) control (pH56.2); (d) control (pH57.2); (^) TMC40 (pH56.2); (m) TMC40(pH57.2); (h) TMC60 (pH56.2); (j) TMC60 (pH57.2) (mean6S.D.; n53).

3.4. Effects on viability of the cells, using typical CLSM pictures of monolayers incubated withpropidium iodide TMC40 and TMC60 are presented. A picture of dead

cells (treated with 0.1% SDS) is given for reasons ofCaco-2 cell monolayers were tested with the comparison. Generally the number of the cells

propidium iodide nucleic stain for their viability after stained by propidium iodide was |5 to 20 cells per4 h of incubation with TMC40 and TMC60 at monolayer for DMEM-HEPES and TMC applica-different concentrations up to 1.0% (w/v). In all tions. This amount of dead cells was less than 0.1%cases the monolayers were able to exclude propidium of the total number of cells that are supposed to beiodide as well and showed no difference with the stained in normal viable cell monolayers [27]. Thiscontrol situation where the monolayers were incu- supports the integrity of the cell monolayers after 4 hbated only with DMEM-HEPES (pH 7.2). In Fig. 5 of application with 1.0% TMC40 and TMC60.

Table 1TEER values (% of initial values) at time 120 min of Caco-2 cell monolayers treated with TMC40 and TMC60 at different concentrations atpH 6.2 and 7.2 (mean6S.D.; n53)

Concentration TMC40 TMC60% (w/v)

(pH56.2) (pH57.2) (pH56.2) (pH57.2)

0.05 47.962.3 50.264.9 61.762.4 61.361.70.1 40.060.8 50.562.7 56.166.0 58.664.20.25 37.362.1 38.962.1 49.663.8 52.763.40.5 37.462.6 30.762.6 46.963.6 44.560.61.0 22.962.0 24.462.0 45.161.05 40.762.6


14Fig. 3. Effect of 0.25% (w/v) TMC 40 and TMC60 on the transport of [ C]-mannitol across Caco-2 cell monolayers at pH values of 6.2and 7.2. (s) control (pH56.2); (d) control (pH57.2); (^) TMC40 (pH56.2); (m) TMC40 (pH57.2); (h) TMC60 (pH56.2); (j)TMC60 (pH57.2) (mean6S.D.; n53).

4. Discussion chitosan is depolymerized at the temperature of608C, yielding polymers of lower number average

Both TMC40 and 60 were synthesized according molar mass (av. M ). But even chitosan salts of lown4to a novel synthesis procedure [24], with which the av. M (8310 D) are not soluble at neutral pHn

control of the degree of substitution of the products values, which indicates the superior solubility of theis possible. The aqueous solutions of both products TMC derivatives. The av. M and the conformationn

showed lower viscosity than the aqueous solutions of in aqueous solutions of these polymers are expectedthe native chitosan salts. An explanation for this to play an important role in their effect on thephenomenon is that during the synthesis the initial permeability of the intestinal tight junctions.

Table 214Apparent permeability coefficients of [ C]-mannitol for cell monolayers treated with pure DMEM-HEPES, TMC40 and TMC60 at different

concentrations at pH 6.2 and 7.2 (mean6S.D.; n53)27 27Concentration TMC40; P 310 TMC60; P 310app app

% (w/v)(pH56.2) (pH57.2) (pH56.2) (pH57.2)

Control 1.160.0 0.760.1 1.160.0 0.760.10.05 5.460.9 7.361.6 14.060.9 18.164.70.1 5.660.7 8.262.0 15.063.6 18.564.10.25 6.160.9 9.160.8 15.560.4 18.964.40.5 10.761.6 17.061.4 17.062.4 23.162.21.0 12.660.4 19.061.7 20.061.7 27.361.6


Fig. 4. Concentration-dependent transport enhancement ratios. (^) TMC40 (pH56.2); (m) TMC40 (pH57.2); (h) TMC60 (pH56.2);(j) TMC60 (pH57.2).

TMC40 was additionally synthesized to determine TMC40 managed to increase the permeability of thethe optimum degree of quaternization on chitosan for radiomarker significantly in a higher degree than atits maximum effect on the permeability of the Caco- pH 6.2. The same effect was observed for TMC60.2 intestinal epithelia. The permeability of the Caco-2 This may be explained by the fact that the Caco-2

14monolayers was investigated using [ C]-mannitol as monolayers, being conditioned for 23 days at mediaa marker for transport of hydrophilic drugs. Mannitol of pH 7.4, showed a stress effect when transferred tois a small metabolically inert molecule, highly medium of pH 6.2, counteracting the effect of thehydrophilic, and it is transported through the aqueous polymers to open the tight junctions and the amountparacellular pathway [27]. of the radiomarker transported. Otherwise, the mech-

Both TMCs managed to decrease the TEER at pH anism of opening the tight junctions under thevalues similar to pH values present in the small influence of positively charged polymers may beintestine [28]. The results show that resistance dependent on the homeostasis of the cell monolayer.measurements are a good indication of the tightness At pH 7.2 TMC60 showed a higher increase in theof the junctions between the epithelial cells, but still permeability of the paracellular marker than TMC40,the resistance is a qualitative parameter. For both which indicates that the extent of the opening of theTMCs and pH values studied (6.2 and 7.2) the tight junctions is strongly dependent on the degree ofdecrease of the TEER was pronounced, indicating quaternization and consequently on the charge den-opening of the tight junctions. Differentiation of this sity of the polymer. It has been proposed thateffect can be observed mainly from the mannitol protonated chitosan (in acidic solutions) can interacttransport profiles, which are a quantitative measure with anionic components (e.g. sialic acid) of thefor the extent of tight junction opening. From the glycoproteins on the surface of the epithelial cells orenhancement ratio data it is obvious that at pH 7.2 the fixed negative charges of the interior of the tight


Fig. 5. Typical CLSM images of the apical side of Caco-2 cell monolayers. (a) Cells treated with 1.0% SDS for 10 min. (b) Cells treatedwith 1.0% TMC40 for 4 h. (c) Cells treated with 1.0% TMC60 for 4 h. Staining was done with the propidium iodide impermeable dead cellprobe.

junctions [30]. It may be possible that TMC is monolayers was negligible using the propidiumtriggering the opening of the tight junctions by a iodide nuclear stain, even when the TMCs weresimilar mechanism such as chitosan salts. tested at relatively high concentrations (1% w/v).

A slight increase of the resistance was observed These findings demonstrate that the mannitol trans-after removal of the polymers from the apical port enhancing effects of TMC40 and TMC60 aresolutions, indicative of the reversibility of this effect. not due to possible cytotoxic activities.Considering the effect of the charge density of theTMCs on the permeability of the Caco-2 mono-layers, the interaction between the apical cell mem- 5. Conclusionsbrane and the TMCs seems to be specific.

It is known that cationic macromolecules like In this study two newly synthesized chitosanpolylysine can exhibit cell toxicity [29]. In the case derivatives were tested concerning their effect on theof TMCs the toxicity found on the Caco-2 cell intestinal permeability of mannitol, and it was shown


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ˇ ˇ[13] J. Kristl, J. Smid-Korbar, E. Struc, M. Schara, H. Rupprecht,cial support of the State Scholarship Foundation of Hydrocolloids and gels of chitosan as drug carriers, Int. J.Greece (I.K.Y.), N.W.O. (The Netherlands), L.U.F. Pharm. 99 (1993) 13–19.

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effect of degree of quaternization of n-trimethyl chitosan chloride for enhanced transport of...

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