!I ELSEVIER
`
`International Journal of Phannaceutics 212 (2001) 19- 28
`
`international
`journal of
`pharmaceutics
`
`www.elscvier.com/locate/ijphann
`
`Novel pH-sensitive citrate cross-linked chitosan film for
`drug controlled release
`
`X.Z. Shu, K.J. Zhu*, Weihong Song
`Department of Polymer Science and Engineering, Zhejiang University, Hang:hou 310027, People's Republic of China
`
`Received 31 July 2000; received in revised fonn 8 September 2000; accepted 8 September 2000
`
`Abstract
`
`Turbidimetric titration revealed that there were electrostatic attractive interactions between citrate and chitosan in
`the pH region of 4.3- 7.6, depending on their degree of ionization. Citrate cross-linked chitosan film was prepared
`simply by dipping chitosan film into sodium citrate solution. The swelling ratio of citrate/chitosan film was sensitive
`to pH, ionic strength etc. Under acidic conditions, citrate/chitosan film swelled and even dissociated in the pH less
`than 3.5, and the model drugs (brilliant blue and riboflavin) incorporated in the film were released quickly (usually
`within 2 h released completely in simulated gastric fluid at 37°C) while under neutral conditions the swelling ratio of
`citrate/chitosan film was less significant and the release rate of brilliant blue and riboflavin was low (less than 40%
`released in simulated intestinal fluid in 24 h). Sodium chloride weakened the electrostatic interaction between citrate
`and chitosan, and therefore facilitated the film swelling and accelerated drug release. The parameters of film
`preparation such as citrate concentration, solution pH etc. influencing the film swelling and drug release profiles were
`examined. The lower concentration and the higher pH of citrate solution resulted in a larger swelling ratio and
`quicker riboflavin release. To improve the drug controlled release properties of citrate/chitosan film, heparin, pectin
`and alginate were further coated on the film surface. Among them only the coating of alginate prolonged riboflavin
`release noticeably (for 80% of drug released the time was extended from 1.5 to 3.5 h with 0.5% w/v alginate used).
`The results indicated that the citrate/chitosan film was useful in drug delivery such as for the site-specific drug
`controlled release in stomach. 0 2001 Elsevier Science B.V. All rights reserved.
`
`Keywords: Chitosan film; Sodium citrate; pH.sensitive; Drug controlled release
`
`1. Introduction
`
`Chitosan with excellent biodegradable and bio(cid:173)
`compatible characteristics is a naturally occurring
`polysaccharide. Due to its unique polymeric
`
`•Corresponding author. Tel.: + 86-571-7952046.
`E-mail address: kjzhu@ipsm.zju.cdu.cn (K.J. Zhu).
`
`cationic character, its gel and film forming prop(cid:173)
`erties, chitosan has been extensively examined in
`the pharmaceutical industry for its potential in the
`development of drug delivery systems (Yao et al.,
`1995; Illum, 1998).
`Chilosan ftlms were usually prepared by chemi(cid:173)
`cal cross-linking with glutaraldehyde etc. (Nakat(cid:173)
`suka and Andrady, 1992; Thacharodi and Rao,
`
`0378·5173/01/$ - see front matter Q 2001 Elsevier Science B.V. All rights reserved.
`Pll: S0378 - 5 l 73(00)00582 -2
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`20
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`X.Z. Shu et af. 1 International Journal of Pharmaceutics 212 (2001) 19- 28
`
`1993; 11lum, 1998). These films swelled under
`acidic conditions due to the ionization of amino
`groups but remained in a shrunken state under
`neutral condition. Moreover, chitosan was re(cid:173)
`ported to have intragastric-floating characteristics
`and prolonged retention of the dosage form in the
`stomach. By utilizing these advantages, chitosan
`films or other dosage forms have been exploited
`widely for oral sustained drug delivery in the
`stomach (Inouye et al.. 1988; Chandy and
`Sharma, 1993; Patel and Amiji. 1996; Gupta and
`Ravi Kumar, 2000). To improve the pH-sensitive
`performance. blended chitosan films have usually
`been prepared. For example, polyether oxide/chi(cid:173)
`tosan film was reported to have excellent pH-sen(cid:173)
`sitivity (Yao et al.. 1993; Angelova et al., 1995;
`Patel and Amiji. 1996).
`However. the chemical cross-linking agents pos(cid:173)
`sibly induce toxicity and other undesirable effects.
`To overcome these disadvantages, recently re(cid:173)
`versible physical cross-linking by electrostatic in(cid:173)
`teraction was applied in
`the preparation of
`chitosan film (Illum, 1998). Polyanions were usu(cid:173)
`ally used as a component to prepare these films.
`For example. Yao et al. (1996) reported the
`preparation of pectin/chitosan films by dissolving
`this polyelectrolyte complex in formic acid and
`then evaporating the solvent. Chu et al. (1995)
`also prepared xanthan/chitosan complex film by
`the solvent evaporation method in the existence of
`concentrated sodium chloride (ca. 0.5 M) and
`then treatment at a high temperature.
`On the other hand, the use of low molecular
`weight ions to prepare an ionic cross-linking poly(cid:173)
`meric matrix was found to be very simple and
`mild, and the cross-linking process was accom(cid:173)
`plished just by dipping the polymer films into
`cross-linking ion solution (Al-Musa ct al., 1999).
`For instance Remufian-L6pez and Bodmeier
`(1997) prepared tripolyphosphate cross-linked chi(cid:173)
`film by dipping chitosan
`film
`into
`tosan
`tripolyphosphate aqueous solution.
`However, up to now. no other anion cross(cid:173)
`linked chitosan film is reported in the literature.
`In our previous experiments, we found that there
`was electrostatic interaction between sodium cit(cid:173)
`rate and chitosan. and citrate cross-linked chi(cid:173)
`tosan beads or microspheres were prepared using
`
`our recently developed method (Shu and Zhu,
`2000a,b). In this paper, we aim to prepare citrate
`cross-linked chitosan film and investigate the pH(cid:173)
`sensitive performances of citrate/chitosan film.
`The preliminary results of citrate/chitosan film as
`pH-dependent drug controlled release matrix are
`also reported.
`
`2. Materials and methods
`
`2.1. Materials
`
`Chitosan was obtained from Tianbao Chitosan
`Co. Ltd (China), and refined twice by dissolving
`in dilute HAc solution and precipitating from
`dilute ammonia, the degree of deacetylation was
`86%, Mv was 460 000. Pectin (USP XXII) and
`sodium alginate (low viscosity) were obtained
`from Sigma (USA). Heparin (Mw 11 000, from
`porcine intestinal mucosa) was a gift from Ji(cid:173)
`uyuan Gene Co. Ltd (China). Riboflavin (Mw
`376.37), theophylline (Mw 180.17) and 5-ftuorou(cid:173)
`racil (5-FU, Mw 130.08) were all purchased from
`Aldrich (USA). Coomassie brilliant blue R250
`(Mw 825) was purchased from Fluka A.G.
`(Switzerland) and used after sieving (less than 50
`µm). Sodium citrate (analytical grade) and other
`reagents were all commercially available and used
`as received.
`
`2.2. Turbidimetric titration
`
`The interactions of sodium citrate and chitosan
`were investigated by turbidimctric titration ac(cid:173)
`cording to the reported method (Park et al., 1992;
`Mattison et al., 1995). A solution of 0.2 g/l
`sodium citrate and 0.2 g/l chitosan was prepared
`at pH 1.0. Titrant (0.01 0.2 M NaOH) was deliv(cid:173)
`ered with a microburette into the solution with
`gentle stirring at 20 ± 0.2°C, and the pH was
`monitored by a digital pH meter with a precision
`of ± 0.01. Changes in turbidity were monitored at
`420 run with an UV vis spectrophotometer and
`reported as 100 - %T which is linearly propor(cid:173)
`tional to the true turbidity for T> 0.9. The lime
`interval between turbidity measurements was ca. 4
`min.
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`X.Z. Shu et al. International Journal of Pharmaceutics 212 (2001) 19- 28
`
`coating of heparin and pectin only retarded drug
`release slightly. The significant difference of cit(cid:173)
`rate/chitosan film swelling and model drug release
`profiles in SIF and SGF indicates that these films
`may be useful for site-specific drug delivery in the
`stomach.
`
`Acknowledgements
`
`This work was supported by National Natural
`Science Foundation of China.
`
`References
`
`Al-Musa, S., Fara, D.A., Badwan, A.A., 1999. Evaluation of
`parameters involved in preparation and release of drug
`loaded in cross-linked matrices of alginate. J. Control.
`Release 57, 223- 232.
`Angelova, N., Manolova, N., Rashkov, I., Maximova, V.,
`Bogdanova, S., Domard, A .• 1995. Preparation and prop(cid:173)
`erties of modified cbitosan films for drug release. J. Bioact.
`Compat. Polyrn. 10, 285- 298.
`Arguelles-Monat, W., Garciga, M., Peniche-Covas, C., 1990.
`Study of the stoichiometric polyelectrolyte complex be(cid:173)
`tween cbitosan and carboxylmethyl cellulose. Polym. Bull.
`23, 307-313.
`Chandy, T., Shanna, C.P., 1993. Chitosan matriA for oral
`sustained delivery of ampicillin. Biomaterials 14, 939- 944.
`Chu, C.·H., Sakiyama, T., Yano, T., 1995. pH-sensitive
`swelling of a polyelectrolyte complex gel prepared from
`xanthan and chitosan. Biosci. Biotech. Biochcm. 59, 717-
`719.
`Dubois, M., Gilles, K.A., Hamilton, J .K., Rebers, P.A.,
`Smith, F., 1956. Colorimetric method for determination of
`sugars and related substances. Anal. Chem. 28, 350 356.
`Gupta, K.C., Ravi Kumar, M.N.V., 2000. Drug release behav(cid:173)
`iors of beads and microgranules of chitosan. Biomaterials
`21, 1115- lll9.
`Tllum, L., 1998. Chitosan and its use as a phannaccutical
`cxcipicnt. Pharrn. Res. 15, 1326- 1331.
`Ikeda, S., Kumagai, H., Sakiyama, T., Chu, C.-H., Naka·
`mura, K., 1995. Method for analyzing pH-sensitive
`swelling of amphotcric hydrogcls -
`application to a
`polyclcctrolyte complex gel prepared from xanthan and
`chitosan. Biosci. Biotech. Biochem. 59, 1422 1427.
`Inouye, K., Machida, Y., Sannan, T., Nagai, T., 1988. Buoy(cid:173)
`ant sustained release tablets based on chitosan. Drug Des.
`Del. 2, 165 175.
`
`Kihuchi, Y., Noda, A., 1976. Polyelectrolyte complexes of
`heparin with chitosan. J. Appl. Polym. Sci. 20, 2561- 2563.
`Macleod, G.S., Collett, J.H., Fell, J.T., 1999. The potential use
`of mixed films of pectin, chitosan and HPMC for bimodal
`drug release. J. Control. Release 58, 303- 310.
`Mattison, K.W., Brittain, 1.J., Dubin, P.L., 1995. Protcin(cid:173)
`polyelectrolyte phase boundaries. Biotechnol. Prog. 11,
`632- 637.
`Nakatsuka, S., Andrady, A.L., 1992. Penneability of vitamin
`B-12 in chitosan membranes. Effect of ctoss-linking and
`blending with poly(vinyl alcohol) on permeability. J. Appl.
`Polym. Sci. 44, 17- 28.
`Park, J.M., Muhoberac, B.B., Dubin, P.L., Xia, J., 1992.
`Effects of protein charge heterogeneity in protein-polyelec(cid:173)
`trolyte complexation. Macromolecules 25, 290-295.
`Patel, V.R., Amiji, M.M., 1996. Preparation of characteriza(cid:173)
`tion of freeze-dried chitosan- poly(ethylene oxide) hy(cid:173)
`drogels for site-specific antibiotic delivery in the stomach.
`Phann. Res. 13, 588- 593.
`Remuiilin-L6pez, C., Bodmeier, R., 1997. Mechanical, water
`uptake and permeability properties of cross-linked chitosan
`glutamate and alginate films. J. Control. Release 44, 215-
`225.
`Shu, X.Z., Zhu, K.J., 2000a. A novel approach to prepare
`tripolyphosphate/chitosan complex beads for controlled
`release drug delivery. Int. J. Phann. 201, 51 - 58.
`Shu, X.Z., Zhu, KJ., 2000b. Chitosan/gelatin microspheres
`prepared by modified emulsification and ionotropic gcla(cid:173)
`tion. J. Microencapsulation, in press.
`Thacharodi, D., Rao, K.P., 1993. Propranolol hydrochloride
`release behavior of cross-linked chitosan membranes. J.
`Chem. Tech. Biotechnol. 58, 177-181.
`Thu, B., Bruheim, P., Espevik, T., Smidsred, 0., Soon-Shiong,
`P., Skjilk-Brmk, G., 1997. Alginate polycation microcap(cid:173)
`sules II. Some functional properties. Biomaterials 17,
`1069-1079.
`Yalpani, M., Hall, L.D .. 1984. Some chemical and analytical
`aspects of polysaccharide modification. 3. Fonnation of
`br.mchcd-chain, soluble chitosan derivatives. Macro(cid:173)
`molecules 17, 272 279.
`Yao, K.D., Peng, T., Goosen, M.F.A., Min, J.M., He, Y.Y.,
`1993. pH-sensitivity of hydrogcl based on complex-forming
`chitosan: polyether intcrpcnetrating polymer network. J.
`Appl. Polym. Sci. 48, 343- 354.
`Yao, K.D., Peng, T., Yin, Y.J., Xu, M.X., 1995. Microcap·
`sulcs/microsphcrcs
`related
`lo chitosan. J.M.S.-REV.
`Macromol. Chem. Phys. C35, 155- 180.
`Yao, K.D., Liu, J., Cheng, G.X., Lu, X.D., Tu, H.L., 1996.
`Swelling behavior of pcctin/chitosan complex films. J.
`Appl. Polym. Sci. 60, 279 283.
`Yoshihisa, T., Yoshioka, I., Segi, N., Ikeda, K., 1991. Acid-in(cid:173)
`duced and calcium-induced gclation of alginic acid: bead
`formation and pH-dependent swelling. Chem. Pharm. Bull.
`39, 1072- 1074.
`
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`
`X.Z. Shu et al.
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`lnternatiunal Juurnal uf PJ1armaceutics 212 (2001) 19- 28
`
`27
`
`However, only the coating of alginate prolonged
`riboflavin release significantly, which indicated
`that there were possibly other reasons but not
`electrostatic interaction resulting in the above re(cid:173)
`sult. It was reported that the aqueous solubility of
`alginate under acidic conditions was very poor
`(Yoshihisa et al., 1991), though in SGF the salt(cid:173)
`bonds between alginate and chitosan dissociated,
`the precipitated alginate layer was still kept on the
`surface of citrate/chitosan film, which may limit
`film swelling and prolong drug release.
`
`3.3.5. Model drug nature
`The model drug properties, especially solubility,
`affected their release behavior from citrate/chi(cid:173)
`tosan film seriously. Fig. 12 shows the loading
`percent and loss percent of brilliant blue, ri(cid:173)
`boflavin, theophylline and 5-FU during cross-
`
`100
`
`101 ~-
`
`10
`
`';!!.
`
`,;::r-
`
`40
`
`10
`
`40
`
`- 201
`-QI
`0
`411 100
`IQ
`10
`QI
`!
`cu
`> ;
`.!! = zo
`e = 0
`
`-•- o
`-
`-025'4
`- 6 - 0,SO'A.
`
`a
`
`- - a
`-o-a.2S'4
`-v-G.10'4
`
`b
`
`r/! ):/==- !
`... l -...,
`--
`••• ~ ::<-..:. •
`~ -
`-•M•
`2:~-
`0
`
`"
`
`1
`
`I
`
`2
`
`j
`
`I 4
`
`I s
`
`c
`I
`
`Time (hours)
`
`Fig. 11. The release of riboflavin from polyanion coating
`citrate.'chitosan film. The films were prepared with 5.0% w/v
`sodium citrate (pH 7.0) and a cross-linking time of I h, and
`then dipped into polyanion solutions (pH 5.5) with different
`concentrations (w/v) for 15 min, (a) pectin, (b) heparin, and (c)
`sodium alginate.
`
`100
`
`o-
`
`e
`
`e
`
`e
`
`e
`
`0
`
`~
`
`l
`]
`
`~
`
`0 brilllllntblue
`rlbollllVln
`lheophytll ..
`ll·FU
`
`4
`v
`
`~~. 0
`'i~t---i
`
`ll
`
`--v
`
`20 ~
`~
`40 c CD
`I!
`60 8.
`80 ~
`100 _.
`
`0.5
`
`1.0
`1.5
`2.0
`Cross-llnklng time (houl'9)
`
`2.5
`
`Fig. 12. The loading percent and loss percent of model drugs
`during cross-linking process, 5.0% w/v sodium citrate, pH 7.0.
`
`linking process. No obvious loss of brilliant blue
`and riboflavin occurred because they slightly dis(cid:173)
`solved in water, and even taking 4 h for cross(cid:173)
`linking the loading percents were both larger than
`97. But for more water-soluble and also smaller
`molecular weight theophylline and 5-FU, the
`loading efficiency decreased greatly with cross(cid:173)
`linking time, and the loading efficiencies were
`both less than 20% in 2.5 h.
`The release of theophylline and 5-FU from
`citrate/chitosan film in SIF was very quick, in
`most cases more than 90% drug released in 2 h,
`while under the same condition the release per(cid:173)
`cents of brilliant blue and riboflavin were both
`less than 5%.
`
`4. Conclusions
`
`Novel citrate cross-linked chitosan film was
`prepared by dipping chitosan film into citrate
`solution. Citrate/chitosan film possessed pH-sensi(cid:173)
`tive swelling and drug controlled release proper(cid:173)
`ties. Sodium chloride weakened ionic cross-linking
`and facilitated film swelling and model drug re(cid:173)
`lease. Sodium citrate solution concentration and
`pH during cross-linking process affected film
`swelling and drug controlled release profiles, and
`using higher concentration and lower pH of
`sodium citrate resulted in less swelling and slower
`drug release. The further coating of alginate on
`the surface of citrate/chitosan prolonged ri(cid:173)
`boflavin release in SGF significantly, while the
`
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`DRL004
`
`

`
`26
`
`X.Z. Shu el al. , lnterna1io11al Journal of Pharmaceulics 212 (2001) 19-28
`
`1.8 I
`a
`__ a
`D
`o
`t$l. - o-o-
`~-A-6-A--------
`1.6 ~arv" - Q-Q-Q
`
`8
`6
`'(/
`
`I
`
`I
`
`2.1 l
`
`2.4
`
`1.4
`
`1.2
`
`a - 1.0 %
`- o - 2.5 %
`- A- 5.0 %
`- <7- 10.0%
`
`~ 1.0c
`g-
`= ! 2.4~ Jl:n.a o
`
`al
`
`b
`
`2 0 1Jrt~ o= :o ===========8
`· ,~ 'e:e=e·======~
`1.s;f
`1.21
`
`a - 1.0 %
`- o - 2.5 %
`- 6-5.D %
`- v - 10.0%
`
`0.8
`0
`
`I
`
`5
`
`I
`
`I
`
`15
`10
`Time (hours)
`
`I
`
`20
`
`I
`
`25
`
`Fig. 8. The influence of sodium citrate concentration on the
`swelling of blank citrate/chitosan film in distilled water (a) and
`SIF (b) (cross-linking time 1.0 h, pH 7.0).
`
`3.3.4. Polyanion coating
`In low pH (1.0-3.5), citrate/chitosan film usu(cid:173)
`ally dissociated and the model drug released
`quickly (Figs. 5 and 6). For example, in SGF the
`release of riboflavin was usually completed within
`2 h. To prolong the drug release from citrate/chi-
`
`6-..__A--6~-.0.
`
`----0---0--0
`---o--a--c
`
`-.
`
`6
`a
`
`-a-pHS.O
`-o-pH6.0
`-.0.-pHT.O
`
`2.8
`
`2.4
`
`2.0
`
`1.6
`
`1.2
`
`.2 -e
`:§
`
`CJ)
`
`1 (/)
`
`.
`0.8
`0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
`Time (hours)
`
`Fig. 9. The swelling curves of blank chitosan film in sodium
`citrate solution (5.0'/a w/v) with different solution pHs during
`cross-linking process.
`
`0
`
`Cll
`
`~
`E 1
`
`CD
`
`1.2
`
`0.9
`
`Cll
`.~ 20
`.... RI
`3
`E 10
`:J
`tJ
`
`11.o.
`
`0
`
`g
`0
`
`-o-pHS.O
`-.O.-pH&.O
`-V-pHT.O
`
`b
`
`~t
`~
`~i"~-----r---r
`/-~~ -
`£_~~
`
`-o-pH 5.0
`-.0.-pH 6.0
`-v-pHT.O
`
`0
`0
`
`5
`
`10
`15
`Time (hours)
`
`20
`
`25
`
`Fig. 10. The swelling of blank citrate/chitosan film (a) and the
`release of riboflavin from citrate/chitosan film (b) in SIF. The
`film was prepared with sodium citrate 5.0% w/v (pH 5.0, 6.0 or
`7 .0) and a cross-linking time of 1.0 h.
`
`tosan film in SGF, polyanions were further coated
`on the surface of citrate/chitosan film. However,
`the coating of pectin and heparin only slightly
`retarded riboflavin release in SGF (Fig. 1 la and
`b). On the other hand, the coating of alginate
`greatly prolonged riboflavin release; the time pe(cid:173)
`riod for 80% riboflavin released was extended
`from 1.5 to 2.4 and 3.5 h after being coated with
`0.25 and 0.50% alginate (w/v), respectively.
`From the point of polyelectrolyte interaction,
`the coating of heparin should retard drug release
`in SGF most effectively, because the interaction
`between heparin and chitosan was the strongest
`due to the highest charge density of heparin (car(cid:173)
`boxylic and sulfonic groups) (Kihuchi and Noda,
`1976). As for pectin and alginate, the weakly
`acidic carboxyl groups protonated in SGF (pH
`1.0- 1.1 ), and the electrostatic attractive force be(cid:173)
`tween pectin (or alginate) and chitosan disap(cid:173)
`peared (Macleod et al., 1999; Yao et al., 1996).
`
`DRL - EXHIBIT 1018
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`

`
`X.Z. Shu et al. t International Journal of Pharmaceutics 212 (2001) 19-28
`
`25
`
`Cl)
`
`-~
`Xi m
`"!
`.~ -.!!
`
`Ill
`
`::I
`E
`::s
`CJ
`
`100
`
`80-l.j.
`60
`
`•
`
`40
`
`20
`
`-•-1.0
`-•-3.5
`-A-4.5
`-0-5,5
`
`-o-&.5
`-A-7.4
`
`-
`
`0
`
`5
`
`15
`10
`Time (hours)
`
`20
`
`I
`
`l: ....
`a
`
`I
`
`25
`
`Fig. 5. The leaching of chitosan from blank citrate/chitosan
`film in buffered solution with the same ionic strength (0.145
`M). The film was prepared with 1.0% (w/v) sodium citrate (pH
`7.0) and a cross-linking time of 1.0 h. pH 1.0 (0.1 M HCI); pH
`3.5, 4.5 and 55 (10 mM acetic acid-sodium acetate buffer);
`pH 6.5, 7.4, 8.5 and 9.5 (10 mM phosphate-buffered solution).
`
`could be seen that in SIF the film swelled to the
`greatest extent at first 2 h, then the swelling ratio
`decreased gradually to a constant value. It was
`probably caused by some further interpolyelec(cid:173)
`trolyte bonds being formed as suggested by Ar(cid:173)
`guelles-Mona} et al. (1990), Macleod et al. (1999).
`In the preparation process, sodium citrate solu(cid:173)
`tion was much in excess and they mainly deter(cid:173)
`mined the preparation pH value in 5.0-7 .0. Fig. 9
`shows the swelling of chitosan film in sodium
`
`•
`
`-•-1.0
`
`-ll.-7.4
`
`-
`*' -; 80
`~
`---<l-55 ~
`~~ ~ l
`.E
`~
`~
`E a a.£.~ 0
`
`a
`
`5
`
`25
`
`Time (hours)
`
`Fig. 6. The release or brilliant blue from citrate/ehitosan film
`in buffered solution with the same ionic strength (0.145 M).
`The film was prepared with 1.0"/o (w/v) sodium citrate (pH 7.0)
`and a cross-linking time of 1.0 h. pH 1.0 (0.1 M HCI); pH 3.5,
`4.5 and 5.5 (10 mM acetic acid- sodium acetate buffer); pH
`6.5, 7.4, 8.5 and 9.5 (JO mM phosphate-buffered solution).
`
`3.0
`
`~ 2.5~ ,,,,..--.-------
`
`g»
`;
`$
`
`U1
`
`2.0 V',V.A-'1_A ____ A -
`~o-o
`1.5
`.(l-C-0-C _____ o - - - -
`c--~
`-c- o
`-o-0.45%
`
`~
`•
`
`a
`
`a
`
`1.0
`
`-~-0.90%
`-v-1.80%
`
`i! 40
`30
`
`Ill
`
`Ill .. ID -; .. QI >
`~ .. '3
`
`§
`
`(J
`
`-c- 0
`-o-0.45%
`-A-0.90%
`-Q'-UO%
`
`,
`
`/Y
`
`-
`
`~~ ~~
`
`20
`
`10
`
`b
`o~~ ........ ~~..-~.....,...~~..--~....,....--1
`10
`25
`15
`5
`0
`20
`Time (hours)
`
`Fig. 7. The swelling of blank citrate/chitosan film (a) and the
`release of riboflavin from citrate/chitosan film (b) in different
`concentration NaCl solution (w/v). The film was prepared with
`5.0% (w/v) sodium citrate, pH 7.0 and a cross-linking time of
`1.0 h.
`
`citrate solution (5.0% w/v) with different pHs
`during cross-linking process. More significant
`swelling occurred in higher pH citrate solution.
`The swelling ratio was 2.51, 2.28 and 2.19 in 4 h
`for pH 7.0, 6.0 and 5.0, respectively. At pH 5.0,
`most of the amine groups of chitosan (more than
`95%) ionized (Fig. 1), so more cross-linking struc(cid:173)
`ture should be formed, which resulted in the
`lowest swelling ratio. However, at pH 6.0 and 7.0,
`only part of amines was ionized (ca. 78% for pH
`6.0 and ca.12% for pH 7.0) and hence less cross(cid:173)
`linking sites formed.
`Fig. 10 shows the swelling and riboflavin release
`from citrate/chitosan film prepared at pH 5.0, 6.0
`and 7.0, respectively, in SIF. In accordance with
`the above discussion, the increase of preparation
`pH resulted in the increase of swelling ratio and
`drug release slightly (Fig. lOa and b). In 24 h,
`with preparation pH 5.0, 6.0 and 7.0, the swelling
`ratio was 1.85, 1.99 and 2.03, respectively, and the
`riboflavin release was 31.1, 34.2 and 39.7%,
`respectively.
`
`DRL - EXHIBIT 1018
`DRL006
`
`

`
`24
`
`X.Z. Shu et al. / International Journal of Pharmaceutics 212 (2001) 19- 28
`
`Fig. 3. The morphologies of bottom surface (a) and cross-section (b) of riboflavin (ca. 20% w/w) loaded citrate/chitosan film. The
`film was prepared with 5.0% w/v sodium citrate (pH 7.0) and a cross-linking time of I h.
`
`in SIF with the cross-linking time extending from
`0.5 to 4.0 h.
`On the other hand, under the same conditions,
`the increase of citrate concentration resulted in a
`decrease of swelling ratio in distilled water (Fig.
`Sa) and in SIF (Fig. 8b} significantly, which indi(cid:173)
`cated that more cross-linking structure formed in
`the case of high concentration of citrate (Remu(cid:173)
`iian-Lopez and Bodmeier, 1997). In Fig. 8b, it
`
`3.0.
`
`0
`
`released for brilliant blue and less than 20% for
`chitosan in 24 h). The results indicated brilliant
`blue release from citrate/chitosan films was
`mainly controlled by the chitosan leaching, i.e. the
`film dissociation.
`
`3.3.2. The salt concentration in media
`Salt usually had a shielding effect on the elec(cid:173)
`trostatic force, and hence weakened the salt-bond
`between citrate and chitosan. Fig. 7 shows the
`influence of sodium chloride concentration on the
`swelling of citrate/chitosan film and the release of
`riboflavin from the film. More significant swelling
`occurred in higher concentrations of sodium chlo(cid:173)
`ride solution, and hence resulted in a quicker
`riboflavin release. In 24 h, with sodium chloride
`concentrations of 0, 0.45, 0.90 and 1.80% (w/v),
`the swelling ratio was 1.67, 2.07, 2.49 and 2.96,
`respectively (Fig. 7a}, and the drug release was
`20.2, 25.8, 27.5 and 39.9%, respectively (Fig. 7b).
`
`3.3.3. Parameters in film preparation
`In our experiments, a cross-linking time of
`more than 0.5 h only slightly limited the swelling
`of citrate/chitosan film and prolonged riboflavin
`release. For example, with 5.0% (w/v} sodium
`citrate (pH 7.0), the equilibrium swelling ratio was
`found to be ca. 1.65 in disti11ed water and ca. 2.0
`
`2.8 I / \
`
`0
`~ I!!
`en
`.5
`m
`~ 2.4
`
`2.6
`
`\ __. ~
`
`-
`
`..!..
`
`4
`
`6
`
`8
`
`10
`
`pH
`
`Fig. 4. The equilibrium swelling ratio of blank citrate/chitosan
`film in buffered solution with the same ionic strength (0.145
`M). The film was prepared with 1.0% (w/v) sodium eitrdte (pH
`7.0) and a cross-linking time of 1.0 h. pH 1.0 (0.1 M HCI); pH
`3.5, 4.5 and 5.5 (10 mM acetic acid sodium acetate bufTer);
`pH 6.5, 7.4, 8.5 and 9.5 (10 mM phosphate-buffered solution).
`
`DRL - EXHIBIT 1018
`DRL007
`
`

`
`X.Z. Shu et al. International Journal of Pharmaceutics 212 (2001) 19- 28
`
`21
`
`Fig. 2. The surface morphology of blank citrate/chitosan film: bottom surface (a) and upper surface (b). The film was prepared with
`S.0% w/v sodium citrate (pH 7.0) and a cross-linking time of 1 h.
`
`3.3. Factors influencing citrate/chitosan film
`swelling and drug controlled release properties
`
`3.3.1. Media pH
`From Fig. 1, it could be seen that the degree of
`ionization of citrate and chitosan was mainly
`controlled by the solution pH; hence citrate/chi(cid:173)
`tosan films exhibited pH-dependent swelling,
`which is shown in Fig. 4. At pH 5.5 and 6.5, the
`swelling ratio was the lowest (2.45- 2.50) due to
`significant electrostatic attraction between citrate
`and chitosan. The decrease of pH weakened salt(cid:173)
`bonds and therefore facilitated the film swelling
`(swelling ratio 2.83 at pH 4.5). Moreover, when
`pH was less than 4.5, citrate/chitosan film swelled
`more significantly and dissociated within 24 h
`because no ionic cross-linking was observed in
`this pH region as revealed by turbidimetric titra(cid:173)
`tion (Fig. I). On the other hand, the increase of
`pH over 6.5 should also weaken salt-bonds, and
`result in a larger swelling ratio (swelling ratio 2.95
`at pH 7.4). However, the further increase of pH to
`8.5 and 9.5 led to the decrease of swelling ratio
`greatly (2.58 and 2.46 for pH 8.5 and 9.5, respec(cid:173)
`tively). It was usually reported that the swelling of
`polyelectrolyte complex films (such as peclin/chi(cid:173)
`tosan film) under weak basic conditions (pH 8-
`10) was very significant (Yao et al., 1996), mainly
`
`resulting from the dissociation of ionic cross-link(cid:173)
`ing and the repelling interaction between nega(cid:173)
`tively charged carboxylic groups. But in our
`experiments, the dissociated citrate at pH 8.5- 9.5
`may diffuse out from the films freely and no
`repelling interaction between carboxylic groups
`existed inside the films, which as well as other
`factors such as the hydrogen-bonding between
`amine of chitosan, attributed to the shrinkage of
`the film in this pH region. Similar results were
`also observed in the case of tripolyphosphate
`cross-linked chitosan film (unpublished results).
`Fig. 5 shows the chitosan leaching from the
`citrate/chitosan film in a buffered solution with
`different pHs at the same ionic strength (0.145
`M). At pH 1.0 and 3.5, the film dissociated
`quickly (within 5.0 h), while at pH 4.5, the leach(cid:173)
`ing percent of chitosan was less than 20% in 24 h
`due to the relatively weak electrostatic attractive
`force between citrate and chitosan (Fig. 1), and at
`pH 5.5, 6.5 and 7.4 no leaching of chitosan
`occurred.
`The release of brilliant blue with poor water
`solubility from citrate/chitosan films also pos(cid:173)
`sessed pH-sensitivity (Fig. 6), which was in accor(cid:173)
`dance with the pH-dependent chitosan leaching
`(Fig. 5) except at pH 4.5 where brilliant blue
`release was faster than chitosan leaching (ca. 67%
`
`DRL - EXHIBIT 1018
`DRL008
`
`

`
`22
`
`X.Z. Shu et al. 1 International Journal of Pharmaceutics 212 (2001) 19- 28
`
`OH
`I
`Hc--c--CH2
`I
`2
`l
`1
`c=o c=o c=o
`_,
`, _
`1-
`0
`0
`0
`
`citrate
`
`Scheme I.
`
`H2N
`
`chitosan
`
`of the same dissolution medium was added back
`to maintain a constant volume. In some cases,
`chitosan leaching from the films was also moni(cid:173)
`tored by the phenol-sulfuric acid color reaction
`(Dubois et al., 1956; Thu et al., 1997).
`
`3. Results and discussion
`
`3.1. The interaction between citrate and chitosan
`
`Citrate is an anion with three carboxylic groups
`and chitosan is a polybase (Scheme 1 ), the charge
`densities of citrate and chitosan are mainly con(cid:173)
`trolled by solution pH (Fig. I). Due to the weak
`acid characteristic of citric acid, under neutral and
`weakly acidic conditions the decrease of the solu(cid:173)
`tion pH resulted in a significant decrease of the
`degree of ionization of citrate. At low pH (less
`than ca. 4.1), the ionization of carboxyl groups
`was normally depressed (the degree of ionization
`
`was usually less than 0.3), i.e. less than one nega(cid:173)
`tive charge was carried by one citrate. For chi(cid:173)
`tosan (a weak polybase), the opposite was the
`case as the ionization of amine groups decreased
`greatly when the solution pH increased above 6.0
`(around the pKa of chitosan 6.3) (Yalpani and
`Hall, 1984), and at pH higher than 7.5 usually less
`than 10% of amine groups were ionized.
`The turbidimetric titration curve of sodium cit(cid:173)
`rate/chitosan is also shown in Fig. l, which is in
`accordance with the pH-dependent charge density
`of citrate and chitosan. At low pH (1.0-4.0), the
`solution was optically clear due to the low charge
`density of the citrate. The turbidity increased
`greatly and the solution began to separate into
`two phases when pH increased over 4.3. This
`could be attributed to the significant charge densi(cid:173)
`ties of citrate and chitosan in this pH region.
`Further increase of solution pH over ca. 6.3 led to
`the decrease of the charge density of chitosan
`greatly, and hence the decrease of turbidity signifi(cid:173)
`cantly. The lowest value of turbidity was observed
`at pH ca. 7.6, and then turbidity increased at pH
`values over 7.6, which was attributed to the poor
`solubility of chitosan in this pH region (Shu and
`Zhu, 2000b ).
`
`3.2. Morphology of citrate/c/1itosanjilms
`
`The surface morphologies of citrate/chitosan
`films are shown in Fig. 2. The bottom surface of
`citrate/chitosan films was very smooth (Fig. 2a)
`while the upper surface was relatively rough (Fig.
`2b ), which was in accordance with the morphol(cid:173)
`ogy of chitosan films before cross-linking (pictures
`not shown). Sodium citrate concentration, pH
`and cross-linking time had little effect on the
`surface morphology of citrate/chitosan films. The
`cross-section of the citrate/chitosan films was very
`integral and dense (pictures not shown).
`The surface and cross-section morphologies
`changed significantly due to the incorporation of
`model drugs into the citrate/chitosan film. For
`example, large pores were observed on both the
`bottom and upper surface of riboflavin loaded
`citrate/chitosan films (Fig. 3a), and the cross-sec(cid:173)
`tion was very rough and many deficiencies were
`observed (Fig. 3b).
`
`1~ c
`0
`~
`M ~
`·2
`
`~6 s -0.4
`
`0
`Cl)
`~
`g>
`0.2
`o.o a
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`pH
`
`Fig. I. The turbidity titration curves of citrate/chitosan solu(cid:173)
`tion at 420 nm (0.2 g/I citrate and chitosan, respcclively), and
`the degree of ionization curves of citrale and chitosan.
`
`50
`
`40
`
`~ 30
`I 8 20
`
`T"
`
`10
`
`0
`
`r(cid:173)
`
`v~
`vX:o,Li.b.4---o.-c.
`v' -·
`. - - ·
`
`-1!!.-cltr.ite
`\
`• \ -v- chltosan
`
`\
`
`1
`
`1.i
`
`~vvvr:-v-v
`
`\'\ "
`/l
`~ .
`·-·-3
`
`DRL - EXHIBIT 1018
`DRL009
`
`

`
`X.Z. Shu el al. 1 International Journal of Pharmaceutics 212 (2001) 19-28
`
`21
`
`2.3. Potentiometric titration
`
`Potentiometric titration was performed accord(cid:173)
`ing to the method reported by Ikeda et al. (1995)
`to evaluate the pH-dependent ionization degree of
`chitosan and citrate, respectively. Sodium citrate
`solution (100 ml; 10 mM) or 0.1% (w/v) chitosan
`solution were neutralized by adding 0.1 M HCl or
`NaOH, respectively, at 20 ± 0.2°C with a mi(cid:173)
`croburette in a nitrogen atmosphere, and the solu(cid:173)
`tion pH was monitored by a digital pH meter with
`a precision of ± 0.01.
`
`2.4. Preparation of cross-linked chitosan film
`
`Chitosan films were produced by a casting/sol(cid:173)
`vent evaporation technique. Chitosan solutions
`(4.0%, w/v) containing model drug (brilliant blue,
`riboflavin, theophylline or 5-FU, 1.0% w/v) were
`prepared by dissolving chitosan and the model
`drug (or dispersing) in 4.0% (w/v) acetic acid. The
`above solutions