壳聚糖-纤维素硫酸钠聚电解质复合物膜对药物表观渗透系数的测试

朱丽英 晏晓琴 张红漫 林东强 姚善泾,* 江 凌

(1南京工业大学理学院,南京211816;2浙江大学化学工程与生物工程系,杭州310027;3南京工业大学食品与轻工学院,南京211816)

1 Introduction

Polyelectrolyte complexes(PECs)are the association complexes formed between oppositely charged macromolecules.These electrostatic interactions are strong enough to prevent dissolution in water and PEC films are capable of maintaining their mechanical strength.1Currently,PECs have been widely used for the preparation of controlled release oral colon-specific drug delivery systems(OCDDS),especially in peptide and protein drug delivery applications.2-4Experiments have shown that PECs can prevent the degradation of peptide and protein drug in stomach and/or small intestine,while they can be degraded by enzymes in the colon.5,6Thus,drug can be released to the site of action in small bowel and/or colon with less gastrointestinal side-effect.Moreover,the technique used for PEC preparation is simple and achieved without the usage of organic acid.7

Recently,we have found that PEC composed of chitosan and NaCS has the potential behavior for controlled release of drugs.8-12As a cationic natural biopolymer,chitosan is chemically a poly-β-(1,4)-D-glucosamine,derived from natural chitin by alkaline deacetylation.Chitosan has been evaluated for conventional and novel gastrointestinal drug delivery systems13,14because of its nontoxic,biocompatible,mucoadhesive,and biodegradable properties.5,15Sodium cellulose sulfate(NaCS)is a hydrophilic cellulose derivative,which has been increasingly used for biomedical application.16-20In aqueous solution,chitosan and NaCS can be combined easily into a water-insoluble polyelectrolyte-complex(chitosan/NaCS complex),since the former is polycation and the latter is polyanion.Wanget al.8prepared a novel colon-specific drug delivery capsule with chitosan and NaCS.Byin vitrodegradation tests,chitosan/NaCS complex could be degraded by colon microflora,and hydrolyzed in the simulated gastric fluid(SGF)and the simulated intestinal fluid(SIF).After that,the chitosan/NaCS complex was extended to form a microcapsules by means of layer-by-layer self assembly with the shell thickness of about 6 nm.9In addition,the degradation of the PEC(chitosan/NaCS complex)by gastrointestinal enzymes was also systematically investigated and the results showed that the degradation could be controlled by varying molecular weight(Mw)of chitosan and NaCS.10So it could be deduced that chitosan/NaCS complex would be a promising material for the oral drug controlled release.

As a potential material for controlled release system,the permeability and swelling properties are essential to determine its characteristicsin vitro.21-23The results of permeability and swelling studies not only reflect the hydration degree and the interaction between polymers in the PEC,but also set references for the further study,e.g.,the use of such polymers for tablet coating.24Generally,the higher swelling ratios would cause the looser network structure of the film,thus leading to high permeability of the drug.

For further investigation of the potential of chitosan/NaCS complex in drug controlled release,we attempt to explore the permeability of drugs through the chitosan/NaCS films.Two substances,including paracetamol(151.16 Da)and 5-aminosalicylic acid(5-ASA)(153.16 Da)would be chosen as the model drugs for testing the permeability of drug across the film.As for the two model drugs,paracetamol and 5-ASA are compounds with similar molecular weight,but the solubility of paracetamol in water(almost 14.3 g·L-1)is much higher than that of 5-ASA(≤1.0 g·L-1).In addition,the acidic strength of 5-ASA is much higher than that of paracetamol.Therefore,the two substances would be used as model drugs with different solubilities and acidic strengths.Additionally,in order to better understand the permeability of the PEC films,the swelling properties will be studied to explore the relationship of the swelling and permeability.Furthermore,the permeability of the films was also evaluated by exposing the films to three simulated solutions,i.e.,the simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and simulated colonic fluid(SCF)forin vitrotests.The investigation would be beneficial to further understand the controlled drug release behavior for chitosan/NaCS complex film or capsule.

2 Experimental

2.1 Materials

Chitosans with an 85%degree of deacetylation and molecular weight of 118.7,135.3,563.3,and 723.2 kDa were supplied by Jinan Haidebei Co.,Ltd.(China).NaCS withMwfrom 5.2 to 710.8 kDa was prepared by heterogeneous reaction as described previously in our laboratory.25Paracetamol and 5-aminosalicylic acid(5-ASA)were purchased from J&K China Chemical Ltd.

The simulated gastric fluid(SGF)(0.1 mol·L-1HCl,pH 1.5;pepsin,12000 U·L-1),the simulated intestinal fluid(SIF)(phosphate buffer solution(PBS),pH 7.4;10000 U·L-1α-amylase,15000 U·L-1trypsin,8400 U·L-1lipase),and the simulated colonic fluid(SCF)(PBS,pH 6.4;cellulase,1200 U·L-1)were prepared forin vitrotests.All chemicals and reagents used were of analytical grade and were used without further purification.

2.2 Preparation of complex film

Chitosan(1%,w/w)and NaCS(4%,w/w)were dissolved separately in 0.1 mol·L-1acetic acid and distilled water,respectively.The chitosan solution was then added to the NaCS aqueous solution slowly and allowed to react for 15 min under mechanical stirring.The dispersions with PEC of NaCS and chitosan were casted onto the Tefloncoated glass plates and dried at 45°C in vacuum.After then,the films were washed with distilled water for three times and dried again.

2.3 Morphology of the complex film

Dried film samples were mounted on brass stubs using carbon paste and the morphology of the complex film was observed on a Hitachi S-4800 scanning electron microscopy(SEM)instrument(Hitachi High-Technologies Corp.,Tokyo,Japan)at required magnification at room temperature.

2.4 Swelling ratio of the PEC Film

The film samples were cut from the bulk film.Samples with thicknesses in the range of 80-120 μm were tested by placing in 0.1 mol·L-1HCl(pH=1.5)and PBS solutions from pH 3.0 to pH 7.4 at(37.0±0.5)°C.The sample masses were accurately determined(±0.0001 g)at 10,30,60,90,120,150,and 180 min after carefully blotting the excessive water on the surface of each sample.The film swelling was expressed using the swelling ratio(SR)that is defined as:

whereW0is the mass of dry sample(g)andWtis mass of sample at timet(g).All experiments were carried out in triplicate.

2.5 Permeability

Drug permeation tests were conducted in Teflon diffusion cells consisting of donor and acceptor compartments(Fig.1).Film samples(circle,diameter of 2.95 cm)were cut with a scalpel and the film thickness was measured at six different places with a micrometer.Samples with mean thickness values in the range of 80-120 μm were selected and mounted between the donor and acceptor compartments.The volume of each compartment was 6.0 mL.Paracetamol and 5-aminosalicylic acid(5-ASA)were used as model drugs(donor)in medium solutions.

The acceptor cells contained blank phosphate buffer solutions.The diffusion cell was placed in a water bath maintained at(37.0±0.5)°C and each compartment was stirred continuously with a magnetic stirrer.At predetermined time intervals,2 mL of medium from acceptor cell was sampled and replaced by an equal volume of fresh medium.The amount of model substances released through the films was determined by spectrophotometer(λ=240 nm for paracetamol,300 nm for 5-ASA)(UV-1601,Shimadzu,Japan).Blank phosphate buffer solutions were used as the control for the measurement of all the samples.Each permeation experiment was repeated three times and the cumulative amount of drug permeated and corrected for the acceptor sample replacement was plotted against time.The permeability coefficients(P)of the various mixed film formulations were calculated using Eq.(2)below:26

where slopeis obtained from the plot of the amount of permeated drug per second(mol·s-1),xis the thickness of the test film(cm),Ais the surface area of the film(cm2),andcdis the drug concentration of the donor solution(mol·mL-1).

Fig.1 Permeation process

2.6 Drug sorption studies

Solutions(0.1 g·L-1)of paracetamol and 5-ASA were prepared with blank phosphate buffer solutions(pH 1.5-7.4).Chitosan/NaCS film samples(1 cm×1 cm)were cut with a scalpel and the film thickness ranged from 80-120 μm.The film samples were added to 5 mL of the drug solutions and shaken in a water bath at(37.0±0.5)°C.Half an hour later,film samples were taken out and the concentrations of paracetamol and 5-ASA in buffer solutions were determined by spectrophotometer(UV-1601,Shimadzu,Japan).

2.7 Statistical analysis

All of the experiments were done in triplicate.Results were expressed as the mean±standard deviation(SD),and differences between two means were considered significantly,on the basis of the Student′sttest,11atp(probability)＜0.05.

3 Results and discussion

3.1 Morphology of chitosan/NaCS films

The morphology of chitosan/NaCS films was investigated from the SEM images.Effects of chitosan/NaCS mass ratios on the morphologies of the complex films were shown in Fig.2.It could be found obviously from Fig.2(a-d)that the surface morphologies of films with 1:2 and 3:4 chitosan/NaCS mass ratios were smoother than the others.When it came to Fig.2(e-h),we could see a clear difference between films with chitosan/NaCS mass ratios at 1:2 and 3:4.When the chitosan/NaCS mass ratio was at 1:2,there were many isolated and irregular micropores(0.1-0.3 μm)in the films.However,while the chitosan/NaCS mass ratio was at 3:4,long and narrow voids were observed in the films,with linear apophyses.This phenomenon could be explained by the formation of the mixed films.The addition of excess chitosan would change the proportions of the crystalline and amorphous regions,and such films would appear to be homogeneous or at least submicroheterogenous.26

3.2 Permeability experiments

Zentneret al.27explained the permeability through polymeric membranes with two mechanisms.One is the‘pore’mechanism whereby drugs are transportedviachannels in the membrane;the other is‘partition’mechanism whereby the drug dissolution in the polymer occurs followed by diffusion along the polymer chains within the membrane.However,the permeation is influenced by a number of factors,including the molecular size of the drug molecule,the state of ionization of the drug molecule at the tested pH,and the interaction between the components within the membrane.16As the films constituted by hydrophilic polymers,the swelling properties directly influence the permeability of the films in aqueous experiments.28So we attempt to correlate the film swelling with permeability data for chitosan/NaCS films.

As shown in Fig.2,the mass ratio of chitosan to NaCS hadgreat influence on the morphology of chitosan/NaCS films.In order to investigate effects of the mass ratio of chitosan to NaCS on the permeability of the complex films,paracetamol was chosen as the model drug.Paracetamol was usually used as a model neutral drug so that variation in drug permeation would not be due to ionization but be attributed to the properties of the films.29Fig.3(a)described effects of the mass ratio of chitosan to NaCS on the permeation behaviors of the complex-films.The lowest and the highest permeation rates were achieved at the mass ratios of chitosan to NaCS of 1:2 and 1:1,respectively.The results were in consistence with the swelling results,since the lowest and the highest swelling ratios were also obtained at the mass ratios of chitosan to NaCS of 1:2 and 1:1,respectively(Fig.3(b)).In addition,the 1:4 and 3:4 samples have the same permeation rate in the first 2 h.The 1:4 sample shows a sudden increase afterwards,while the 3:4 sample remains the same rate.3:4 was close to the optimum ratio when NaCS and chitosan were tightly combined.However,1:4 was the ratio that excess NaCS existed and was entwined by chitosan.After 2 h in the solution,the film swelled,the structure became looser,and the pores were bigger.As a result,the 1:4 sample shows a sudden increase afterwards,while the 3:4 sample remains the same rate.It was reported that higher swelling occurs when fewer ionic crosslinks are present in the film.28Thus the results in Fig.3 indicated that the tightest structure of chitosan/NaCS complex film would be expected to occur near the chitosan/NaCS ratio of 1:2.So film samples in the following experiments were prepared with the chitosan/NaCS ratio.

Fig.2 Morphologies of chitosan/NaCS films

For further study of the permeation properties of chitosan/NaCS complex-films,two substances were selected as the model drugs,including paracetamol and 5-ASA.Paracetamol and 5-ASA were employed to investigate the influence of solubility in water and acidic strength of drugs on the permeability.

Effects of theMwof chitosan and NaCS on the permeability of chitosan/NaCS films are shown in Fig.4.It was obvious that the swelling results and permeability of chitosan/NaCS films were greatly influenced by theMwof chitosan and NaCS.Enormous differences were found in the swelling ratios of films prepared with chitosan of 118.7 and 135.3 kDa,as well as the films based on NaCS of 5.2 and 31.2 kDa.This may be attributed to the specificity of each other′sMwin the formation of PEC films.As shown in Fig.4(a),both the permeability constants ofparacetamol and 5-ASA were in consistent with the trends of the swelling ratios.The higher swelling ratio caused the higher permeability constant.In addition,the permeability constants of 5-ASA were higher than the ones observed with paracetamol.This result might be caused by the affinity of the substances to the components of the PEC films,26and the hydrogen bond interaction involved.Since the acidic strength of 5-ASA is much higher than that of paracetamol,5-ASA tend to be ionized in acidic solutions.As a result,5-ASA would be attracted to the film.Unlike the results in Fig.4(a),Fig.4(b)showed that the films formed by lowMwNaCS did not obey the role:high swelling ratio leads to high permeability.The reason lies in the fact that NaCS of lowMwis easier to dissolve in water than that of highMw(≥169.7 kDa).As a result,drugs would be easy to be soluble in the films and to pass through the films.

Fig.3 Effects of chitosan/NaCS mass ratio on the swelling properties and permeability of chitosan/NaCS films

Fig.4 Effects of the Mwof chitosan and NaCS on the permeability of chitosan/NaCS films for paracetamol and 5-ASA

Fig.5 Effects of pH on the permeability of chitosan/NaCS films

Fig.5 described the variation of permeability constants and swelling ratios of paracetamol and 5-ASA at different pH.It was obvious that the permeability results of 5-ASA were not in consistent with the swelling tests and the permeation behavior of paracetamol.As shown in Fig.5,the permeability constant of paracetamol decreased with the increase of pH,and reached the minimum value at pH 5.0.Further increase of pH resulted in the increase of permeability constants.However,the variation of the permeability of 5-ASA did not agree with the swelling results,a quite high permeability constant was obtained at pH 3.0,in spite of the low swelling ratio.The phenomenon may be explained from the charge state of 5-ASA and the complex film.5-ASA would carry negatively charged carboxyl group at pH 3.0,since its pKais 2.3 and 5.69.The complex films would carry positively charge at pH 3.0,for the isoelectric point is about pH 5.0.Therefore,5-ASA would be electrostatically adsorbed onto chitosan/NaCS films at pH 3.0,compared with other pH.The presumption was confirmed by drug sorption studies(data not shown).At pH 3.0,about 5.0×10-4g 5-ASA was adsorbed by 1.0 g chitosan/NaCS films,however,no apparent decrease of 5-ASA concentration was observed in other solutions.For paracetamol,it was a neutral drug,and it would carry weak or no charge in the solution.No evident loss of paracetamol was found in the tested solutions.Accordingly,the variation in the drug permeation would not be due to ionization,but be attributed to the physicochemical properties of the films and the changes they undergo in the diffusion media.28Therefore,the permeation of paracetamol directly reflected the swelling properties of the complex-films.

3.3 In vitro test

In order to investigate deeply the behaviors of chitosan/NaCS complex films in controlled drug release,the complex films were put in three simulate solutions,i.e.,SGF,SIF,and SCF,in sequence with gastrointestinal enzymes(Table 1).According to the above results,paracetamol was chosen as the tested drug because it is a neutral drug.As can be seen in Table 1,the formulations 3,4,5 exhibited a higher permeability after 3 h in SGF with enzyme than that without enzyme which was shown in Fig.4(1.5×1010cm2·s-1).After permeation for 3 h,about 30%paracetamol from the donor compartment would pass through the films.Moreover,when the three samples were put in SIF for 6 h,the samples were disintegrated.As for formulations 2 and 6,they showed relatively low permeability coefficients in SGF,but performed relatively high permeability after 6 h in SIF.After 3 h in SGF and 6 h in SIF,about 50%paracetamol would permeate the complex-films.Different from the above samples,formulations 1 and 7 showed lower permeability coefficients after degradation in SGF and SIF,and the amount of permeated paracetamol was less than 40%.The rea-son may lies in the fact that the two films showed relatively low swelling ratios.The pores in the films were small,thus making the enzymes inaccessible to the films or blocked in the films.As a result,the permeation capability of paracetamol was highly decreased.Therefore,the PEC of chitosan and NaCS showed the potential for oral gastrointestinal drug delivery.

Table 1 Permeability coefficients for chitosan/NaCS films with different formulations through simulated gastrointestinal tract

4 Conclusions

The permeabilities of drugs across chitosan/NaCS films were investigated by varying chitosan/NaCS mass ratios and theMwof chitosan and NaCS.The results showed the permeability was closely related with swelling results.Besides,theMwof chitosan and NaCS had great influence on the permeability of the PEC films.When it came to the types of drugs,the permeation rate of charged drug,5-ASA,was higher than that of paracetamol,and the proteins were easier to pass through the films with lowMwNaCS.In vitrotests showed that chitosan/NaCS films had great potential for gastric,intestinal,and colonic targeted release.

(1) Berger,J.;Reist,M.;Mayer,J.M.;Felt,O.;Gurny,R.Eur.J.Pharm.Biopharm.2004,57,35.doi:10.1016/S0939-6411(03)00160-7

(2) Park,M.R.;Chun,C.;Cho,C.S.;Song,S.C.Eur.J.Pharm.Biopharm.2010,76,179.doi:10.1016/j.ejpb.2010.06.012

(3)Van Der Gucht,J.;Spruijt,E.;Lemmers,M.;Stuart,M.J.Colloid Interface Sci.2011,361,407.doi:10.1016/j.jcis.2011.05.080

(4)Assaad,E.;Blemur,L.;Lessard,M.;Mateescu,M.A.;Biomat,J.Sci.Polym.E2012,23,1713.

(5) Xia,W.S.;Liu,P.;Liu,J.Bioresour.Technol.2008,99,6751.doi:10.1016/j.biortech.2008.01.011

(6)Chen,C.;Liu,M.Z.;Lu,S.Y.;Gao,C.M.;Chen,J.C.;Biomat,J.Sci.Polym.E2012,23,2007.

(7) Shu,X.Z.;Zhu,K.J.Int.J.Pharm.2002,233,217.doi:10.1016/S0378-5173(01)00943-7

(8) Wang,M.J.;Xie,Y.L.;Zheng,Q.D.;Yao,S.J.Ind.Eng.Chem.Res.2009,48,5276.doi:10.1021/ie801295y

(9) Xie,Y.L.;Wang,M.J.;Yao,S.J.Langmuir2009,25,8999.doi:10.1021/la9014338

(10) Zhu,L.Y.;Lin,D.Q.;Yao,S.J.Carbohyd.Polym.2010,82,323.doi:10.1016/j.carbpol.2010.04.062

(11) Wu,Q.X.;Zhang,Q.L.;Lin,D.Q.;Yao,S.J.Int.J.Pharm.2013,455,124.doi:10.1016/j.ijpharm.2013.07.048

(12) Wu,Q.X.;Yao,S.J.Colloid Surface B2013,109,147.doi:10.1016/j.colsurfb.2013.03.035

(13) Hejazi,R.;Amiji,M.J.Control.Release2003,89,151.doi:10.1016/S0168-3659(03)00126-3

(14) Tozaki,H.;Komoike,J.;Tada,C.;Maruyama,T.;Terabe,A.;Suzuki,T.;Yamamoto,A.;Muranishi,S.J.Pharm.Sci.1997,86,1016.

(15) Onishi,H.;Machida,Y.Biomaterials1999,20,175.doi:10.1016/S0142-9612(98)00159-8

(16) Chen,G.;Yao,S.J.;Guan,Y.X.;Lin,D.Q.Colloid.Surface B2005,45,136.doi:10.1016/j.colsurfb.2005.08.002

(17) Zhang,J.;Yao,S.J.;Guan,Y.X.J.Membrane Sci.2005,255,89.doi:10.1016/j.memsci.2005.01.025

(18) Zhang,J.;Guan,Y.X.;Ji,Z.;Yao,S.J.J.Membrane Sci.2006,277,270.doi:10.1016/j.memsci.2005.10.038

(19) Zhao,Y.N.;Chen,G.;Yao,S.J.Biochem.Eng.J.2006,32,93.doi:10.1016/j.bej.2006.09.007

(20)Fluri,D.A.;Kemmer,C.;Baba,M.D.E.;Fussenegger,M.J.Control.Release2008,131,211.doi:10.1016/j.jconrel.2008.07.036

(21) Van den Mooter,G.;Samyn,C.;Kinget,R.Pharm.Res.1994,11,1737.doi:10.1023/A:1018911316021

(22) Macleod,G.S.;Collett,J.H.;Fell,J.T.J.Control.Release1999,58,303.

(23)Akhgari,A.;Farahmand,F.;Garekani,H.A.;Sadeghi,F.;Vandamme,T.F.Eur.J.Pharm.Sci.2006,28,307.doi:10.1016/j.ejps.2006.03.005

(24) Macleod,G.S.;Fell,J.T.;Collett,J.H.Int.J.Pharm.1999,188,11.

(25) Yao,S.J.Chem.Eng.J.2000,78,199.doi:10.1016/S1385-8947(00)00131-5

(26) Donbrow,M.;Friedman,M.J.Pharm.Pharmacol.1975,27,633.doi:10.1111/jphp.1975.27.issue-9

(27) Zentner,G.M.;Cardinal,J.R.;Kim,S.W.J.Pharm.Sci.1978,67,1352.doi:10.1002/jps.v67:10

(28) Miller,M.D.;Bruening,M.L.Chem.Mater.2005,17,5375.doi:10.1021/cm0512225

(29) Ofori-Kwakye,K.;Fell,J.T.Int.J.Pharm.2001,226,139.doi:10.1016/S0378-5173(01)00802-X