Novel PH-sensitive Chitosan-based Hydrogel for Encapsulating Poorly Water-soluble Drugs

Novel pH-sensitive chitosan-based hydrogel for encapsulating poorly water-soluble drugs Tse-Ying Liu * , Yi-Ling Lin Institute of Biomedical Engineering, National Yang-Ming University, 155, Sec. 2, Lih-Nong St., Taipei, Taiwan, ROC a r t i c l e i n f o Article history: Received 22 April 2009 Received in revised form 31 August 2009 Accepted 5 October 2009 Available online 9 October 2009 Keywords: Chitosan derivatives pH-sensitive hydrogel Cell compatibility Controlled release a b
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  Novel pH-sensitive chitosan-based hydrogel for encapsulating poorlywater-soluble drugs Tse-Ying Liu * , Yi-Ling Lin Institute of Biomedical Engineering, National Yang-Ming University, 155, Sec. 2, Lih-Nong St., Taipei, Taiwan, ROC  a r t i c l e i n f o  Article history: Received 22 April 2009Received in revised form 31 August 2009Accepted 5 October 2009Available online 9 October 2009 Keywords: Chitosan derivativespH-sensitive hydrogelCell compatibilityControlled release a b s t r a c t Carboxymethyl–hexanoyl chitosan (CHC) is an amphiphilic chitosan derivative with excellent swellingability and water solubility under natural conditions. In this work, the influence of the degree of carboxy-methyl and hexanoyl substitution on the pH-sensitive swelling behavior, drug release behavior, andantiadhesion behavior of CHC hydrogels (cross-linked with genipin) were studied. It was found thatthe pH sensitivity was more pronounced in CHC than in N,O-carboxymethyl chitosan because the hexa-noyl group altered the state of water in CHC by inhibiting intermolecular hydrogen bonding. In addition,greater pH sensitivity was observed in samples bearing longer hydrophobic chains (carboxymethyl–palmityl chitosan). Interestingly, when used with ibuprofen (a poorly water-soluble therapeutic agentused here as a model drug), the bursting release of the drug was less prominent in the CHC sampleshaving a high degree of carboxymethyl substitution. The CHC hydrogel also demonstrated good cell com-patibility and its antiadhesive ability after grafting was altered by changes in the degree of hexanoylsubstitution.   2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction In recent years, considerable attention has been focused onchitosan (CS) hydrogels and their use in tissue engineering scaf-folds, controlled release and implants [1–3]. This is because of theirglycosaminoglycan-like structure and wide range of outstandingcharacteristics such as biodegradability and availability. However,their application is limited by their poor water solubility underneutral physiological conditions, poor solubility in organic sol-vents, and lack of amphipathicity. Moreover, it is known that an in-crease in the hydrophobicity of a drug-loaded hydrogel, whenadministered via the mucosal route, will not only improve drugencapsulation efficiency but also drug transport across the buccalmucosa [4,5]. Therefore, several chitosan derivatives have beendeveloped over the years with improved properties for enhancedapplicability [6–8].Recently, our group developed a novel chitosan derivative (car-boxymethyl–hexanoyl chitosan, CHC) with excellent water solubil-ity under neutral conditions [9]. In addition, we found that thepresence of both carboxymethyl (hydrophilic) and hexanoyl(hydrophobic) groups affords an amphiphilic nature, which mightmake CHC suitable for use as a drug-loaded implant material forpoorly water-soluble agents. In an earlier report, it was suggestedthat a CHC monolithic drug-loaded membrane could efficientlyencapsulate ibuprofen (a nonsteroidal anti-inflammatory drug,IBU) which is poorly water soluble in the neutral physiologicalenvironment. In addition, recently, CHC micelles were also suc-cessfully prepared and employedfor encapsulation of an antitumoragent with poor water solubility [10]. Moreover, it is expected thatthe CHC hydrogel might exhibit pH-sensitive behavior since itbears both acidic (COOH) and basic (NH 2 ) functional groups. Theeffects of the ionic functional groups on the nature of the pHresponse of chitosan derivatives have been described by severalresearchers [6,11]. However, little research has been done on theinfluence of hydrophobic substitution groups on this pH response.In our previous study, we performed preliminary investigations onthe water absorption and water retention behavior of CHC [9].However, it remains unclear whether the ligand substitutionsinfluence the pH sensitivity of the resulting amphipathic hydrogel.In general, the swelling (water absorption) behavior of pH-sen-sitive hydrogels is determined by the ionization of the functionalgroups of hydrogel and the intermolecular volume for water; thelatter depends on the macromolecular structure, the state of water,the hydrophobic/hydrophilic characteristics, and the electriccharge [12–14]. It is known that these factors also may govern celladhesion to materials as well as drug release [15–17]. Hence, theinfluence of the hydrophobic substitution groups on the celladhesion and drug release behaviors of the CHC hydrogel were alsoinvestigated in this study. The CHC hydrogel has a hyaluronan-likestructure (Scheme 1) with controlled hydrophilicity/hydrophobic-ity and therefore has the potential to be employed as an 1742-7061/$ - see front matter    2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.actbio.2009.10.010 *  Corresponding author. Tel.: +886 2 28267923; fax: +886 2 28210847. E-mail address: (T.-Y. Liu).Acta Biomaterialia 6 (2010) 1423–1429 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage:  ntiadhesive membrane encapsulating therapeutically active agentsthat prevent postoperative tissue adhesion.The present study is focused on the influence of the degree of carboxymethyl and hexanoyl substitution on the pH-sensitiveswelling behavior. In addition, IBU was employed as a poorlywater-soluble model drug in order to investigate drug releasebehavior. Furthermore, a preliminary investigation of the variationin cytotoxicity and antiadhesive properties with the degree of hexanoyl substitution was also carried out. 2. Experimental  2.1. Materials Chitosan ( M  w  = 215,000 g mol  1 , deacetylation degree = 80%,insoluble impurity < 1%), hexanoic anhydride, palmitic anhydride,and ibuprofen were purchased from Sigma–Aldrich. Genipin wassourced from WAKO. Methanol was purchased from TEDIA.  2.2. Synthesis of CHC and carboxymethyl–palmityl chitosan (CPC) CHC was synthesized from N,O-carboxymethyl chitosan(NOCC). The synthesis of NOCC and CHC with various degrees of substitution has been reported in our previous work [9]. In brief,the home-made NOCC with low and high degrees of carboxy-methyl substitution were named NOCC-1 and NOCC-2, respec-tively. The NOCC samples (2 g) were dissolved in distilled (DI)water (50 ml) and stirred for 24 h. The resulting solutions weremixed with methanol (50 ml), followed by the addition of hexanoicanhydride at concentrations of 0.3 M (low degree of hexanoylsubstitution) and 0.5 M (high degree of hexanoyl substitution).CPC, an amphiphilic NOCC derivative bearing a longer hydrophobicchain, was produced by using palmitic anhydride instead of hexa-noyl anhydride. After a reaction time of 12 h, the resulting solu-tions were dialyzed against an ethanol solution (25% v/v) for24 h. The obtained ethanol/water-soluble (volume ratio = 3:2)chitosan derivatives with various degrees of carboxymethyl, hexa-noyl, and palmityl substitution were named as shown in Table 1.For the subsequent material characterization, a 1.3% (w/v) solutionof each chitosan derivative was prepared by dissolving the ob-tained derivatives in DI water. In order to prepare hydrogels, thesesolutions were then cross-linked with genipin solution (1% w/v;molecular structure shown in Scheme 2a) at 50   C for 2 days[18]. The molar ratio of genipin to chitosan derivative was fixedat 300 for all samples to obtain the hydrogels with identicalcross-linking density (i.e. effective number of cross-links per unitvolume) rather than identical cross-linking degree (i.e. effectivenumber of cross-links/total number of the  D -glucosamine residuesavailable for cross-link), which means that the influence of theretractile force on the swelling behavior was almost equal for eachderivative. This is beneficial to enhance and clarify the influencesof carboxymethyl and hexanoyl groups on the pH-sensitive swell-ing ratios and drug release behaviors of the CHC hydrogels.  2.3. Material characterization Proton nuclear magnetic resonance ( 1 H NMR) spectra were re-corded by an NMR spectrometer (Varian UNITYINOVA 500) at270 MHz to confirm the degree of substitution. Attenuated totalreflectance–Fourier transform infrared (ATR–FTIR) spectra wererecorded on a spectrometer (Bomem DA8.3, Canada) using afilm-type sample (4 cm  0.5 cm). The ATR–FTIR spectra were re-corded at a resolution of 2 cm  1 in the range 4000–400 cm  1 .The state of water was characterized by differential scanning calo-rimetry (DSC; Perkin-Elmer Instruments) [19]. Each dried samplewas weighed in an aluminum pan to which different amounts of DI water were then added. Prior to the DSC test, samples with var-ious water absorption ratios ( W  C ;  W  C  =  W  w / W  d , where  W  w  and  W  d are the weights of the moist and dry samples, respectively) werequenched from room temperature to   60   C and conditioned atthe same temperature for 10 min. The DSC curves were then ob-tained by reheating to 300 K at a scanning rate of 10 K min  1 .  2.4. Characterization of the swelling ratio The samples for the swelling test were dried in a vacuum cham-ber with P 2 O 5  for 24 h prior to the experiment. The test was Scheme 1.  Molecular structures of CHC.  Table 1 Estimated substitution degree (by  1 H NMR) for each sample. Carboxymethyl group Hexanoyl group  D -Glucosamine residue Degree of substitution NOCC-1 0.32 0 0.75CHC-1A 0.32 0.26 0.49CHC-1B 0.32 0.46 0.29NOCC-2 0.50 0 0.73CHC-2A 0.50 0.26 0.47CHC-2B 0.50 0.48 0.25Palmityl groupCPC 0.50 0.40 0.33Degree of N-acetyl- D -glucosamine for all samples was around 0.2. Scheme 2.  Molecular structures of (a) genipin and (b) ibuprofen.1424  T.-Y. Liu, Y.-L. Lin/Acta Biomaterialia 6 (2010) 1423–1429  performed by immersing each sample into solutions of various pH(pH 1–10) at 20   C for 48 h. The swelling ratio ( W  S ) at equilibriumwas determined by Eq. (1): W  S  ¼ð W  wet  W  dry Þ = W  dry  ð 1 Þ where  W  wet  and  W  dry  are the weights of the sample after and beforethe swelling test.  2.5. Cytotoxicity and cell adhesion tests Cytotoxicity tests were performed by the elution methodaccording to ISO 10993-5. Fibroblast cells (L929) were culturedin Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fe-tal bovine serum and were plated at a density of 2  10 5 cells ml  1 in a 24-well plate at 37   C in 5% CO 2  atmosphere. After 24 h of cul-ture, the medium was replaced with extract fluids obtained byplacing the CHC hydrogel (0.2 g ml  1 of culture medium) in the cellculture medium at 37   C for 24 h. The cells grown in the culturemedium and in that containing 5% dimethylsulphoxide (DMSO)under the same conditions for 24 h acted as negative and positivecontrols, respectively. The adherent cells were trypsinized, centri-fuged, and resuspended for vital cell counting using a hemacytom-eter combinedwith an inverted phase-contrast microscope. For thecell adhesion test, fibroblast cultures were prepared from themeninges covering the brains of Wistar rats that were purchasedfrom the Animal Center, National Taiwan University (Taipei, Tai-wan). All animals used in the present study were anesthetizedand sacrificed in strict adherence to guidelines from the Guidefor the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1985) under a protocol ap-proved by the Animal Center Committee of National Defense Med-ical Center. Cells were seeded at 5  10 5 cells ml  1 onto six-wellplates coated with the CHC hydrogel. Cells cultured on chitosan-coated plates under the same conditions acted as controls. After24 h of culture, the adherent cells were observed using an invertedphase-contrast microscope.  2.6. Drug encapsulation efficiency and release test  IBUwasemployedasapartiallyhydrophobicmodeldrug(molec-ular structure shown in Scheme 2b). IBU-loaded monolithic films(matrix films) were prepared from the chitosan derivatives usingthe film-casting method. In brief, a chitosan/acetic acid solution,NOCC/DIwatersolutions,andCHC/ethanol/DIwatersolutionsweretaken and their pH values were adjusted to 7.0 using a 1 M NaOHsolution. These polymeric solutions were then mixed with IBU toform IBU-loaded polymeric solutions (IBU concentration =1.2 mg ml  1 ). Subsequently, the drug-loaded polymeric solutionswerepouredintoPetridishesandcross-linkedwithgenipintoformdrug-loaded matrix films after drying at 50   C for 2 days. To calcu-late the real encapsulation efficiency, the cross-linked matrix filmswere rinsed with an ethanol solution for 30 s and the amount of IBU rinsed out into the ethanol solution ( L 2 ) was determined byUV–visible spectroscopy (Agilent 8453) at 264.4 nm using a prede-terminedstandardconcentration–intensitycurve.TheIBUencapsu-lation efficiency ( E  ) was determined by Eq. (2): E  ¼ð L 1  L 2 Þ = L 1  100 %  ð 2 Þ where  L 1  is the initial loading amount of IBU incorporated. The IBUrelease test was performed by placing the above-mentioned IBU-loaded membrane (  50 mg) in a quartz cuvette containinga releasemedium (50 ml) with constant rotary shaking at 37   C. The releasemedium (phosphate-buffered solution; pH 7.4) was withdrawn atspecific time intervals and replaced with an equivalent volume of fresh buffer. The drug release profile was obtained by plotting acurve of   M  t  / M   against  t  , where  M  t   is the amount of drug releasedat time  t   and  M   is the amount of drug released once the equilibriumstate is reached. 3. Results and discussion  3.1. Synthesis of CHC  CHC was synthesized from NOCC. Upon ligand substitution, ahydrogen atom of the amino group was replaced by a hexanoylgroup. The molecular structure is shown in Scheme 1 and thedegree of substitution confirmed by  1 H NMR spectra is shown inTable 1.  3.2. pH-sensitive swelling behavior  The influence of the carboxymethyl group on the pH sensitivityis shown in Fig. 1. Both NOCC-1 and NOCC-2 showed similar pHsensitivity with a transition (i.e. the point with the minimumswelling ratio) in the range pH 7–8; this is thought to be due toelectrostatic attraction between the COO  and NH 3 þ groups. Belowand above the transition point, the swelling ratio increased due tothe electrostatic repulsion between the identical NH 3 þ groups ð NH 3 þ . . . NH 3 þ Þ and the COO  groups (COO  . . . COO  ), respectively.The influence of the hexanoyl group on the swelling ratio at vari-ous pH was also investigated. For the CHC and NOCC samples withthe identical degree of carboxymethyl substitution, such as CHC-1A and NOCC-1, since the number of   D -glucosamine residues avail-able for cross-link of the CHC samples were reduced by hexanoylsubstitution, it is reasonable to believe that the cross-linking de-gree of CHC-1A was supposed to be higher than that of NOCC-1while the cross-linking density was fixed. Interestingly, as shownin Fig. 1, the swelling ratios of CHC-1A and CHC-2A were higherthan those of NOCC-1 and NOCC-2, respectively. This implies thatthe roles of carboxymethyl and hexanoyl substitutions on theswelling behaviors of the chitosan derivatives employed in thepresent work need to be further explored. In general, besides theconsideration of cross-linking degree and cross-linking density,the swelling behavior is determined by intermolecular interactionssuch as hydrogen bonding, hydrophobic interaction, and electro-static interaction, which depend on the macromolecular structureand the state of water. Therefore, the influence of hexanoyl substi-tution on the macromolecular structure and the state of water inthe chitosan derivatives were characterized by ATR–FTIR and DSC. Fig. 1.  Influence of carboxymethyl and hexanoyl substitution on the swelling ratioat various pH. T.-Y. Liu, Y.-L. Lin/Acta Biomaterialia 6 (2010) 1423–1429  1425  The ATR–FTIR spectra are shown in Fig. 2. The characteristicpeaks of amide I bands (1635 cm  1 ) of NOCC-1 and NOCC-2 exhib-ited a significant red shift ( d  = 20 cm  1 ) compared with those of unmodified chitosan, which can be attributed to the fact that theintermolecular hydrogen bonds in NOCC (O @ CNH 2 . . . O @ COH) arestronger than those in unmodified chitosan (amide resonanceH-bonding, O @ CNH 2 . . . O @ CNH 2 ). This is because the dipole mo-ment of an N A H bond is smaller than that of an O A H bond. How-ever, the extent of the above-mentioned red shift decreased ashexanoyl groups were introduced (CHC-1A and CHC-2A). More-over, the characteristic peaks assigned to the carboxyl dimer(O @ COH . . . O @ COH, at 1723 cm  1 ) observed for the CHC sampleswere broader then those observed for the NOCC samples, whichimplies that the carboxyl groups in CHC are in the form of amonomer (O @ COH) rather than a dimer. These findings suggestthat the formation of intermolecular hydrogen bonding (i.e.O @ CNH 2 . . . O @ COH and/or O @ COH . . . O @ COH) in the CHC samplesmight be inhibited by hexanoyl substitution to a certain extent. If true, it would be reasonable to believe that CHC should have a dif-ferent state of water than NOCC. As evidenced in Fig. 3a, all the DSCendothermic peaks were noticeably lower than 4.8   C, indicatingthat free water was not present in the NOCC-2 hydrogel even inthe fully swollen state ( W  C  = 2, which is almost equal to the  W  S  va-lue of NOCC-2) [19,20]. However, as shown in Fig. 3b, peak III of  CHC-2A was very close to 4.8   C, indicating that the CHC-2A hydro-gel contains free water when not fully swollen. This, together withthe ATR–FTIR observations, demonstrates that the polymer–poly-mer interaction was weakened and the volume of intermolecularspace for free water increased on the introduction of hexanoylgroups. Thus, the swelling ratios of the CHC samples were signifi-cantly higher than those of the NOCC samples. Furthermore, oncethe formation of intermolecular H-bonding (i.e. O @ CNH 2 . . . O @ COHand/or O @ COH . . . O @ COH) was inhibited by hexanoyl substitution,the electrostatic repulsion  ð NH 3 þ . . . NH 3 þ and COO  . . . COO  Þ probably became dominant among the intermolecular interactionswhen the pH was less than the p K  a  of COO  and larger then the p K  a of NH 3 þ . Furthermore, the electrostatic repulsion force did notseem to be inhibited on hexanoyl substitution, probably becauseelectrostatic interactions form under different conditions thanhydrogen bonding interactions; the latter require a specificdistance and angle. Therefore, a more prominent level of pH sensi-tivity was observed in the CHC samples than in the NOCC samples,although they had the same degree of carboxymethyl substitution.If the above argument is correct, it is then possible that the above-mentioned phenomenon (i.e. the pH sensitivity being enhanced bythe presence of hexanoyl groups) will become more pronouncedwhen a longer side chain (CPC) is introduced. As evidenced inFig. 4, the swelling ratios of CPC at low and high pH were consid-erably greater than for CHC-2A, which agrees with the abovehypothesis.  3.3. In vitro cytotoxicity and cell adhesion test  CHC might serve an antiadhesion function due to its hyaluro-nan-like structure, thus making CHC a candidate implant materialwith the potential for preventing postoperative tissue adhesion[21,22]. Therefore, the in vitro cytotoxicity and antiadhesive prop-erties of CHC were investigated in the present study. The cytotox-icity of the newly synthesized CHC is shown in Fig. 5. Nostatistically significant difference ( P   > 0.05) in the population of surviving cells was detected between the negative controls andthe CHC samples. In addition, significant differences ( P   < 0.05) incell survival were observed between all samples and the positivecontrols. In these preliminary tests, CHC did not show any signsof cytotoxicity in vitro and the degree of hexanoyl substitutiondid not affect the number of surviving cells. The influence of thedegree of hexanoyl substitution on cell adhesion was also studied.In general, hydrophobicity/hydrophilicity, electric charge, surfacemorphology, and the nature of surface functional groups are Fig. 2.  ATR–FTIR spectra of chitosan derivatives. Fig. 3.  DSC curves of the chitosan derivatives (carboxymethyl substitutiondegree = 0.5) measured at  W  C  = 2 under neutral conditions. The dashed linesrepresent the fitted curve (Lorentzian curve-fitting). Fig. 4.  Swelling ratios of CHC-2A and CPC at various pH, recorded at 37   C.1426  T.-Y. Liu, Y.-L. Lin/Acta Biomaterialia 6 (2010) 1423–1429
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