Full Paper

pH- and Temperature-Sensitive Chitosan
Hydrogels: Swelling and MRI Studies
Francisco M. Goycoolea, Marı´a E. Ferna´ndez-Valle, Inmaculada Aranaz,
´ ngeles Heras*
A

MRI and swelling experiments are used to probe the state of water and infer the microstructure of chitosan hydrogels. SEM reveals a porous open scaffold-type structure for hydrogels
that were equilibrated at 2 8C before freezing as compared to those equilibrated at 37 8C. ADC
MRI measurements reveal an anisotropy in
the microstructure of these gels. T1 relaxation
MRI values were larger as the pH increased
from 7.6 to 12.0, the result of a lower rate of
exchange between protons of the hydration
sphere of the polymer and bulk water. The
thermosensitive and pH-sensitive properties
of these hydrogels can be utilized in the development of innovative materials for biotechnological and biomedical applications,
including criobiocatalysis and bioremediation
as well as in programmed drug delivery.

F. M. Goycoolea, M. E. Ferna´ndez-Valle, I. Aranaz, A´. Heras
Instituto de Estudios Biofuncionales, Departamento de Quı´mica
Fı´sica II, Facultad de Farmacia, Universidad Complutense, Paseo
de Juan XXIII, 1, 28040 Madrid, Spain
Fax: þ34 91 394 3284; E-mail: aheras@farm.ucm.es
F. M. Goycoolea
Laboratory of Biopolymers, CIAD, AC. Carr. a La Victoria Km 0.6
P.O. Box 1735, Hermosillo, Sonora, 83000, Mexico
F. M. Goycoolea
Current address: Institute of Plant Biology and Biotechnology,
Westphalian Wilhelm’s University of Mu
¨nster, Hindenburgplatz
55, 48143 Mu
¨nster, Germany
M. E. Ferna´ndez-Valle
Current address: CAI de RMN, Instituto Pluridisciplinar, Paseo de
Juan XXIII No. 1, 28040 Madrid, Spain
I. Aranaz
Current address: Instituto de Ciencias y Tecnologı´a de Polı´meros
de Madrid, CSIC, Juan de la Cierza 3, 28006 Madrid, Spain
´ . Heras
A
Instituto de Estudios Biofuncionales, Departamento de Quı´mica
Fı´sica II, Facultad de Farmacia, Universidad Complutense de
Madrid, Paseo de Juan XXIII 1, 28040, Spain
Macromol. Chem. Phys. 2011, 212, 887–895
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction
Over the recent decade or so, increasing interest has been
focused on employing natural polymers such as polysaccharides, namely cellulose, starch, alginate and chitosan, as
well as proteins such as gelatin, to design and engineer
hydrogels with a specific response to a physicochemical or
biological environment. Chitosan is particularly attractive
in this context, due to its biodegradability, biocompatibility, low toxicity, wound healing and antimicrobial
activity that make it a very interesting polymer for the
development and engineering of materials to be used in
food, biomedical, pharmaceutical, cosmetic and biotechnological applications including membrane technology,
bioremediation, biosensors and biocatalysis.
Due to the poor mechanical strength of chitosan itself,
crosslinking is needed to harness biomaterials with
improved strength and elasticity that allow processing it

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DOI: 10.1002/macp.201000301

887

This information has allowed to anticipate potential future fields of applications for these materials.[23.[12] textile industry. Once the excess of alkali is removed. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co. Experimental Part Materials Chitin (DA ¼ 79 mol-% as determined by solid-state CP-MAS 13 C NMR spectroscopy) from prawn shell waste (Pleoticus mu ¨elleri) was kindly donated by Dr.[18–19] the gel materials obtained from alkali chitin addressed in the present study do not require chemical derivatization and their networks are thought to be governed by physical interactions. To this end.0–8.[16] Also. Hydrogels have been studied by using this technique with different purposes such as study of drug release from hydrogels[20] study of fixation of hydrogel scaffolds into a defect site for tissue engineering. Universidad Nacional del Sur. cosmetic.7 – 2. All standard chemicals were USP grade. Chem. While MRI measurements have allowed to measure the apparent diffusion coefficient (ADC). the glass plate was removed and the gels were expelled by gentle pressure from a pipette applied on the rim of each cylinder in the plate. The perforated plate was placed gently on top of the alkali chitin solution allowing filling the cylinders uniformly.[15–17] By contrast with other previous works that have aimed to develop pH sensitive hydrogels based on chitosan.[5] hydrogen-bonding.[10–11] drug delivery. the resulting hydrogels are found to be formed by chitosan (1. Goycoolea. To rid the gels from NaOH. Alkali chitin itself has lower critical solution temperature (LCST) at 30 8C. 212. Spain). in previous studies.[25–28] To the best of our knowledge. tissue enginerring. we have addressed the thermal and mechanical properties of chitosan hydrogels as a response 888 to pH and temperature.7 mm) according with the procedure described elsewhere.[1–4] ionotropic.0.[17] These gels are obtained as a result of a phase separation concomitant to de-acetylation of alkali chitin. by the conversion of chitin into chitosan.[14] The gels were prepared using a homemade polytetrafluoroethylene (PTFE) plate (70  70 mm2) perforated with 225 cylinders of welldefined dimensions (diameter 3 mm. we have conducted detailed swelling studies at varying pH and temperature and have complemented them with SEM and MRI imaging techniques to glean further understanding of the mechanisms that control the behavior of these systems as a response to changes in their microenvironment. to be a very powerful tool in the study of slow diffusion processes in polysaccharide gels. films. E. l ¼ 8. A www. under vacuum this temperature has been found to decrease. the gelling process is accompanied by substantial deacetylation of chitin and hence. Silicon grease avoided leakage. from Panreac (Barcelona. corresponding to the exact volume to fill the perforated cylinders in the plate (13. in previous studies. this is the first study that accounts for the use of MRI techniques to characterize the physicochemical properties of chitosan hydrogels.mcp-journal. and allows obtaining cross-sectional or 3D images of the solid materials and living organisms in a non-destructive way.5 wt.[9] Chitosan hydrogels have sucessfully been used in food. sponges. Preparation of Chitosan Hydrogels Chitin (1. Aranaz.MaterialsViews.[16] Once washed to neutrality.-%) added with ice according with the protocol of Sannan et al. Magnetic resonance imaging (MRI) is a well-known imaging technique widely applied in medicine and biology.8 mL). thus allowing to probe the state of water within the gel networks and infer aspects of the microstructure and properties of these systems. 2011. The gels were collected on a water bath kept at 65 8C under Macromol. Heras F.[17] In the present study. The loaded plate was left to stand under vacuum at 25 8C for 72 h. to design molecularly imprinted materials. gellan and agarose. was placed on a piece of glass at the center of a square made with silicon grease corresponding with the rim of the plate. Ferna´ndez-Valle. chitosan hydrogels obtained under such protocol present a volume phase transition at 20 8C that is observed only under a narrow range of pH 7.com . M. driven by standing under vacuum at 25 8C for 72 h. we have characterized the swelling response at varying pH and as a result of stepwise changes in temperature.[15] however.[6] hydrophobic association. namely by covalent. KGaA.24] Pulsed-field gradient magnetic resonance techniques using strong field gradients (either in NMR or MRI) has proven. Argentina). membranes. a process that inexorably led to setting of turbid hydrogels molded as cylindrical rods. Weinheim www.´ . I. their diffusion and relaxation behavior and relate these to their swelling properties at varying pH. dextran. Often these materials are based on chitosan gels that at molecular scales can be envisaged as three-dimensional macromolecular networks.[7–8] or a combination of the last two along with crystallite formation. in particular. beads or fibers. including alginate. Viviana Ramos (LIBAQ. M. Chitosan hydrogels are crosslinked gel networks obtained in aqueous conditions by virtue of various crosslinking mechanisms. we have demonstrated that chitosan hydrogels prepared from alkali chitin solutions prepared at low temperature (0 8C).[29]An aliquot of alkali chitin.-%) was fully dissolved in aqueous NaOH (16 wt. and the proton relaxation T1 and T2 parameters for the gels at varying pH.de into scaffolds.[15–17] In either case.[14] set as a result of a phase separation process effected either by heating[15] or by applying vacuum at room temperature. scanning electron microscopy (SEM) has allowed to image the surface morphology of the hydrogels in their most pronounced swollen and collapsed states. Phys. A preliminary account of the swelling behavior of these gels has been published elsewhere.6 wt.[21] water transport properties.[13] In recent studies.[22] dynamic swelling.% with a high degree of acetylation (DA  27–41%) and Mw varying in the range 52–71 kDa.

8). T1 and T2 and the standard deviation over four ROI that represents all the area of the gel on the image. Phys. However. T2 map was calculated from a series of spin-echo images with different echo times.7 T. Swelling Studies The degree of swelling of freeze-dried gels was examined by immersing the gels in Eppendorf vials containing 1. The uptake of solvent was monitored by periodic withdrawing of the gel cylinders from the vials and accurately weighing them after removal of the excess surface solvent by light blotting with a filter paper. Two different experiments with diffusion gradients applied along two different directions were acquired.5 cm the slice thickness (SLTH) was 1. For these experiments only one coronal slice was acquired.5 ms. . Fresh gels had an average polymer concentration of 2. The relative swelling ratio (S) was determined gravimetrically and represented the gel solvent uptake by S¼ wh ws ws (1) where wh is the weight of the hydrated gel and ws is the weight of the dried gel. JSM 6400). Hydrogels were mounted on a brass mount and sputtered with Au/Pd in a Balzers SCD 004 Sputter Coater. the MRI results are bound to reflect the state of the gels at low temperature (2 8C).0 mm and the matrix size was 256  256. . Chem. SLTH ¼ 1. Scanning Electron Microscopy (SEM) Surface morphology of freeze-dried hydrogels was determined using a scanning electron microscope (JEOL.MaterialsViews.0 at 25 8C is illustrated in Figure 1. After equilibration at one temperature.5 mL of buffer solutions of pH varying between 6. Germany) imaging spectrometer operating a 4.com Swelling of polymer hydrogels can be seen as two distinct mass transfer steps: the first consists of the solvent convection through the pores and the second one in the diffusion of the solvent into the polymer network struts. as the gels www. Ettlingen. KGaA. For the ADC measurements a spin-echo diffusion weighted sequence was used.5) adding KCl as needed. A total of 11 images with recovery times between 15 and 2 000 ms were acquired. A portion of the hydrogels was kept fresh for MRI measurements. while another one was frozen by rapid immersion in liquid nitrogen and freeze-dried for swelling experiments.6)  (7.5 cm.5 cm and SLTH ¼ 1. Further exhaustive washings were conducted at room temperature. FOV ¼ 1. The apparent diffusion coefficient (D) was calculated from a series of diffusion experiment weighted images at different diffusion gradient strength. the gels were then re-equilibrated at a different temperature. Weinheim 889 . TR ¼ 2 000 ms. echo train length ¼ 4. MRI experimental measurements were conducted at room temperature as our system did not allow for temperature control. T1 relaxation time measurements were conducted using an inversion-recovery scheme combined with a fast spin-echo (FSE) sequence. number of accumulations ¼ 2. T1 map was calculated from a series of inversion recovery fast spin-echo images with different inversion times.0 mm. The influence of pH on Seq values followed the trend (12. were kept immersed at the various pHs at 2 8C and taken out immediately before loading them into the NMR capillary tubes. Clearly a pH-dependent behavior is observed for both the evolution of solvent uptake and the overall degree of swelling at the point at which little further swelling is observed (>80 h).0 (citric acid/Na2HPO4) or in alkali solution of pH ¼ 12.de magnetic stirring. FOV ¼ 1. The degree of acetylation of the chitosan was 27% as measured by firstderivative UV spectroscopy[30] in freeze-dried gels.d. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co. The selected slice was the same one than that for the calculation of T1 relaxation time. From these parametric maps we obtained the mean values for D. Results and Discussion Swelling Studies MRI studies Swelling Kinetics at Varying pH at 25 8C Instrumentation All MRI experiments were performed using a BIOSPEC BMT 47/40 (Bruker. Germany). The vials were immersed in a circulating water bath equilibrated to the desired temperature. www. 212.8–8. The acquisition parameters of the images were: time between gradients (D) ¼ 20. The three tubes were placed in a home-built solenoidal coil (i. TE ¼ 45 ms. For T2 relaxation time measurements 20 echoes of three coronal slices using a spin-echo (CPMG) sequence were acquired. For these experiments the repetition time (TR) was 3 000 ms. Computing The parametric maps were calculated using the Image Sequence Analysis (ISA) utility of the ParaVision 3. ¼ 5 mm) truncated from the top. Individual hydrogel samples previously left to equilibrate at 2 8C in buffer or alkali solutions at varying pH were loaded (without solvent) in three separate standard NMR tubes (i. Measurements were conducted in triplicate. Ettlingen.-%.mcp-journal. by changing the water of the beaker several times and until complete removal of NaOH was ensured from pH measurements.0 mm and matrix size ¼ 256  256.0 (NaOH/KCl) at isoionic strength (I ¼ 0. the effective TE was 35 ms. The cylinders weight was monitored individually until the hydrated weight reached a constant value. ¼ 15 mm) and the probe head was placed at the center of the magnet.3) > (7. Each diffusion experiment consisted on six diffusion weightings with b values between 136 and 5 656 s  mm2. The swelling kinetics of freeze-dried chitosan gels at varying pH in the range 7.3 to 12.0) > (7. echo times (TE) values were 16 or 30 ms the field of view (FOV) was 1. 2011.1 program (Bruker. These were regarded as the pseudoequilibrium swelling values (Seq).0. The parameters for the FSE image were: TR ¼ 4 000 ms.6 wt.and Temperature-Sensitive Chitosan Hydrogels . In all Macromol. time of pulse gradient (d) ¼ 10 ms. Magnetic field gradients were generated by a 12 cm actively shielded gradient set.pH.d.

82  0. l ¼ 8.3 1. hence D was the only floating parameter to fit. Ferna´ndez-Valle. (2) Swelling Behavior at Varying pH during Stepwise Changes in Temperature where C represents the concentration within the hydrogel at time t and location x and D is the diffusion coefficient.3 pH 7.de Table 1.6 1. M. Evolution of the relative swelling degree of chitosan hydrogel cylinders (diameter 3 mm. The values of ADC swelling thus obtained.´ . Weinheim www. 212.6 experienced a two-fold increase in   1 X S 4 Da2n t ¼ 1 exp  Seq a2 r2 n¼1 n (3) 25 25°C 2°C 25°C 20 S (swelling ratio) where an is the value of the Bessel function for each term n (n ¼ 16). Chem.75  0.22 8. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co.0 1.0 pH 12. Note that at pH ¼ 7. l ¼ 8. the greater ADC swelling values were registered for the gels at pH ¼ 7. Although only a very small fraction of –NHþ 3 groups are likely to survive in the protonated form in chitosan at pH ¼ 7.com . E. a Levenberg-Marquardt non-linear least squares minimization routine in Origin 7. Effect of pH on the swelling parameters of chitosan hydrogels at 25 8C.15 12. are shown in Table 1.6–8.0 4 2 0 20 40 60 80 100 120 Time (min) Figure 1. 14 S (swelling ratio) 12 pH 8 6 pH 7. thus effectively reflecting that the velocity of water uptake during the swelling process is favored at pHs  7. This is ascribed to the fact that at 25 8C. Evolution of the relative swelling degree of chitosan hydrogel cylinders (diameter 3 mm.[15–17] In turn. I. Heras F. r is the radius of the cylinder and t is time.3.6 pH 8.0.3 and 12. In order to solve equation 3 and compute the ADC swelling values. The fit was performed using the actual r values measured directly from the corresponding MRI parametric diffusion maps (see below) and it was assumed to remain unchanged.33  0. Seq. this seems to be enough so as to modify profoundly the swelling and diffusion properties of these systems as it is further discussed in this and related papers.6 pH 8.0 1.7 mm) at 25 8C and varying pH (as shown in label). along with those of swelling equilibrium. D is the apparent diffusion coefficient (ADC swelling).mcp-journal.33  1. The solution of Fick’s equation for cylinder symmetry is given as An experiment was devised to evaluate the change of swelling behavior during stepwise change in temperature under the cycle 25 ! 2 ! 25 8C. according to Fick’s diffusion equation[31]  2  @C @ C ¼D @t @x2 7.43 8. cases it was possible to model the solvent diffusion process as a one-dimensional process where diffusion in the gel cylinders is considered everywhere radial.MaterialsViews.18 12.6 and 7. Macromol.5 was used.0 the gels attain the lower Seq values as compared to gels at pH ¼ 7. M.6.0 5 0 0 1000 2000 3000 4000 5000 6000 7000 Time (min) Figure 2. due to the predominance of hydrophobic hydration favored by the almost full neutralization of –NHþ 3 groups in chitosan.93 pH ¼ 7. Phys.7 mm) at varying pH (as shown in label) and during stepwise changes in temperature under the cycle 25 ! 2 ! 25 8C. Aranaz.32 10. Indeed.0 are in a collapsed state.15 7. the gel networks in the vicinity of 890 Equilibrium swelling Seq 109 m2  s1 10 0 ADC swelling 15 10 pH 7. KGaA. 2011.6.0 pH 12.6–8. A www. Please note in Figure 2 that the hydrogels at pH ¼ 7. Goycoolea. within this narrow range of pH chitosan hydrogels have been found to exhibit their maximum thermal sensitivity.13 8.

6.0 may lead to the destruction of hydrophobic hydration and hence. when the gels are left to swell to equilibrium directly at 37 8C. at pH ¼ 8.0 are in agreement with those registered for chitosan hydrogels crosslinked at their surface with glutaraldehyde. By contrast. whereby after application of the stimulus the gel can be loaded with the enzyme.3–7. maximum and minimum swelling are attained at pH ¼ 7. Phys.6 as the gels exhibit positive thermosensitivity. the gels immersed at pH ¼ 12 did not show any temperature-dependent behavior in their swelling kinetics as their S values increased monotonically with time throughout the experiment. the greatest swelling degree is achieved at pH ¼ 7. 2011. 25 and 37 8C as well as for gels that had been first equilibrated first at 37 8C and then transferred to 2 8C. On heating back to 25 8C. The greater ionization of –NHþ 3 at pH ¼ 7. gels attain collapsed and swollen states respectively at 2 and 25 8C. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co. This property could be exploited in some specific fields of application. when the hydrogels are equilibrated at 25 8C. The existence of this transition can account for the behavior of the gels addressed in this work at the vicinity of pH ¼ 7. it is likely that only a very little amount of protonated –NHþ 3 groups exist. experience a dramatic increase in Seq with a clear maximum observed yet again at pH ¼ 7. Yet. as hydrophobic hydration by water protons bound to the polymer (i. Inspection of the data reveals that when the gels are left to hydrate directly at 2 8C.3–12.de their swelling state from those initially attained at 25 8C upon changing the temperature to 2 8C.6. this fact seems to induce an optimal balance between hydrophilic and hydrophobic character and hence the gel network experiences the greatest negative thermosensitivity evidenced by swelling at low temperature and deswelling at high temperature.6 could be exploited in enzyme and biomacromolecules immobilization. i.6. for the gels attain swollen and collapsed states at 2 and 25 8C.0. At 25 8C. In turn. it is very interesting to notice that the system behaves almost in direct contrast with the profile exhibited at 2 8C and respectively. very large swelling is observed at pH ¼ 6. In previous studies. Weinheim 891 . the gels at this pH deswelled reversibly to the same S value previously attained during the first swelling step. In turn. and after enzyme deactivation the matrix can be unloaded and thereafter used again.6. as water ‘‘cages’’) is bound to lead to greater association between polymer chains as the temperature increases. Equilibrium swelling for freeze-dried chitosan hydrogels as a function of pH and temperature (as shown in label). .6. KGaA. Macromol. e. the behavior is completely opposite to that observed at pH ¼ 7.g.[32] It is very interesting to notice that at pH ¼ 12 chitosan hydrogels experience virtually no dependence in their swelling behavior between 2 8C and 37 8C.and Temperature-Sensitive Chitosan Hydrogels .8 but at any greater pH. the swelling degree decreases sharply and in the vicinity of pH  7.6 and decreases sharply at lower or greater pH values.6 attains a minimum.3–7.com dominated by a fine interplay between hydrophilic forces given by the state of ionization of chitosan and hydrophobic hydration of chitosan. we argue that at a given temperature and pH the behavior of the gel system is Figure 3. presumably because the gel is already above its LCST and thus in its collapsed state. To account for these results. respectively. where the role of the partial protonation of amino groups in chitosan in the properties of the gels could be studied. The thermal sensitivity exhibited by chitosan hydrogels at pH ¼ 7. .6 exhibit a phase transition centered at  20 8C as evidenced by micro-DSC and smalldeformation rheology. In turn.[17] The transition enthalpy of these hydrogels attains its maximum at the vicinity of pH ¼ 7. Meanwhile. This behavior is clearly enhanced when the gels are previously hydrated at 37 8C and further cooled to 2 8C.pH. gels that were left firstly to equilibrate at 37 8C and then cooled and left to equilibrate at 2 8C. 212.[29] At this pH. When equilibrated directly at 37 8C.mcp-journal. hydrogen bonding and other weak interaction forces that are destroyed at high temperature are favored and may account for the positive thermal sensitivity. pH has no influence on Seq. Figure 3 shows swelling data of chitosan gels equilibrated directly at 2. the gels showed only a slight increase in their swelling when cooled to 2 8C but did not deswell upon heating back to 25 8C.e. The pH and thermal sensitivity of the gel swelling behavior was further explored in a series of experiments in which the lower limit of pH was extended to 6. At pH ¼ 7.MaterialsViews. presumably as a consequence of the polymer network relaxation previous to cooling.0. there is virtually no pH dependence on swelling and the overall Seq values are slightly greater than those at 25 8C in the range of pH ¼ 7. however.8.e. it has been possible to demonstrate that chitosan hydrogels at pH ¼ 7.0–7. bioremediation. as in a recently described approach for hydrogels of PNIPA comprising PEG to control the network mesh size. Chem. www. the results at pH ¼ 7. www.

719  0. all differences in swelling degree were observed.0 5 790  65. it seems reasonable to argue that contrast. Goycoolea.25 15.de These values were calculated along the radial and axial directions to afford ADC MRI radial and ADC MRI axial. At this pH.5 8.62  1.667  0.0 1. I. reveals a non porous water.MaterialsViews. 212. as they are not too far after being equilibrated at 2 8C.01  0. swelling experiments and confirm that these gel materials leading to overall greater ADC MRI values than those at at pH  7. pH ADC MRI radial ADC MRI axial T1 T2 109 m2  s1 109 m2  s1 ms ms 7. Interestingly. chitosan is bound to be fully ‘‘closed’’ states can be controlled by a change in temperature.e.09 21.6 equilibrated at 2 and at the gels. Notice that at each pH condition both ADC MRI values are not the same.00004 1 687. Heras F. the at 2 8C. A www. These results seem to suggest that water diffuses through the gel network slightly more rapidly along the axial than along the Figure 4.5  6.91 17.780  0. swelling. while no hydration water is observed.01  109 m2  s1.922  0. MRI measurements were conducted at room temperature in the absence of buffer solutions for hydrogels previously equilibrated overnight at 2 8C. neutralized and hence to confer a more hydrophobic character to the gel network.6 – water a) 892 2. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co.6 at 2 8C (left panel) and radial direction. but to some extent also at pH ¼ 7.[26] Macromol. To understand the diffusion behavior of chitosan Diffusion Behavior at Varying pH hydrogels at varying pH.0. These results were consistent with the previous thickness of the hydration shell is expected to be reduced. However.15  3.9 2 180. SEM images recorded for chitosan hydrogels at pH ¼ 7. with interconnected pores of average diameter  5 mm. Indeed.0. Phys. Both techniques probe the The morphology and ultrastructure of the surface of freezediffusion of bulk water through the molecular mesh of dried chitosan hydrogels at pH ¼ 7.6 behave as gating devices whose ‘‘open’’ and pH ¼ 8.0 46. at their greatest degree of from the apparent diffusion coefficient of pure water.02 a) 2.00002 1 800.´ . Effect of pH on the apparent diffusion coefficient (ADC MRI). Chem.46  2.0.02 b Equilibrium swelling.6 and 12. Bar represents 60 mm. KGaA.0 1.01  0. Ferna´ndez-Valle.708  0.6 or 12.2  8.00005 1.6 and 8. as under such conditions the greater found at pH ¼ 12.0 were of the same order of magnitude as the ADC Scanning Electron Microscopy swelling values (Table 1).00003 1.0 the ADC MRI radial values are greater than the ADC MRI axial ones. we suggest that it is the increase in the void Table 2. a number of factors must be In Table 2 are shown the computed ADC MRI values for the considered.com . overall MRI Studies smaller ADC MRI values). particularly at pH ¼ 12.610  0. as ‘‘closed’’ rough topology. to notice that in general the ADC MRI values obtained for the gels at pH ¼ 7.2  11. completely different to that observed chitosan is ionized in the gel network at pH ¼ 7. is characterized by an open scaffold-type structure 2. 2011.e. By From these results.0 (1. Aranaz. It was also interesting 37 8C (right panel).00003 1 580. the largest ADC MRI values were 37 8C was investigated. It can readily be the estimated ADC MRI values are likely to be related appreciated in Figure 4 that the microsctructure of the gels to the rapidly diffusing water.94  109 m2  s1). resulting in a larger hydration shell where mobility of water is restricted (i.mcp-journal. respectively.6 1. In general.9 65. i.0 58. is concerned. Weinheim www. As far as the rapidly diffusing water component chitosan hydrogels at the three studied pH conditions.5 12. most of the attenuated measured MRI signal is due to bulk corresponding to the collapsed state. the structure of the gels equilibrated at 37 8C. E. T1 and T2 relaxation MRI parameters of chitosan hydrogels equilibrated at 2 8C by MRI (mean average values of four analyzed ROIs). M. M.0  5. Seq Cited values.00002 1.

e. Phys.e. at pH ¼ 7. This is a behavior that may underlie the development of very interesting future applications.MaterialsViews. will not any longer be able to exchange with water at pHs above their pKa. Inspection of the data reveals that the elevation of the pH of the hydrogels from 7. unless there is efficient magnetization transfer between the polymer and water protons in the hydrogels. there is a concomitant disruption of the hydrophobic hydration cluster around acetyl groups in chitosan. The anisotropy observed in the diffusion behavior (ADC MRI radial versus axial values) could stem on the molecular organization of chitosan during the set up of the gels whereby oriented structures are formed within the gels network at the mesoscale. in dangling ends. Our MRI instrument did not allow Macromol. were well represented by single inversion recovery curves (results not shown) can be taken as an indication that non-exchangeable polymer protons made no contribution to the relaxation process. polymer protons) would have decayed substantially during the time delay (35 ms) between the excitation 908 pulse and the commencement of signal acquisition. hence contributing to a decrease in T1.0. as the hydroxyl groups pKa is  11. associated with the sol fraction.[34] However. –CH. KGaA. At this high pH.6 to 12. namely. The obtained results seem to indicate that there is a reduction in the net amount of bound water at the polymer at alkaline pH. In addition. The fact that T1 data for the gels at the three studied pHs addressed in this work. The relaxation results obtained from MRI measurements for the gel series at varying pH are shown in Table 2. partial ionization of –OH groups is expected (i. on loosing their proton. leading to an increase in the net amount of structured water and hence reducing the mobility of bulk water around water cage-like hydrophobic hydration sites. 2011. Such processes can be associated.0. larger pores) within the gel network what leads to large ADC MRI radial values in the gels at pH ¼ 12.com highly dependent on pH). thus.6. T1 and T2 are sensitive to molecular motions of very different timescales. it seems clear that the variation in the ionization state of the chitosan chains determines the overall degree of solvation and mobility of rapidly and slowly diffusing water protons through the polymer gel network via a fine hydrophilic/hydrophobic balance. thus effectively increasing T1. Seq. respectively. particularly in the biomedical field where chitosan hydrogels can be applied as mimics of sctructures of the extracellular matrix in connective tissues. and interjunctions) will tend to average out dipolar interactions and hence they will not couple together. side chains.1 to 7. with the coupling of proton magnetization between water and the polymer through chemical exchange driven by a change in ionization of hydroxyl exchangeable protons groups in the polysaccharide (i. In turn. . Two parallel effects could account for this.[35] and later applied to develop a model for polysaccharide gels using high-resolution NMR.pH. 212. pKa of chitosan is  6. gel water protons and exchangeable protons with relatively short T2. a greater amount of free water.de volume (i.5).e. two opposing contributions to relaxation are expected from water-rich hydrogels. bound to be slow enough and of restricted enough amplitude so as to couple together and communicate to each other via spin diffusion due to strong dipolar interactions. junction zones). the small proportion of surviving protonated –NHþ 3 groups will account for the new increase in ADC MRI radial values. and to a less extent with non exchangeable coupled polymer protons (–CH3. This progressive increase observed on T1 as the pH ascends could be the result of a lower rate of exchange between protons of the hydration sphere of the polymer and bulk water and ‘‘through-space’’ cross-relaxation processes. C6 at acetyl and at the sugar ring. respectively). www.[36–37] It assumes that the various types of water protons each have a different average state of binding to the polymer. Therefore. This might be due to the fact that the signal from any short T2 components (e. A theory proposed to characterize in a quantifiable manner the contribution of multiple proton species with exchange of magnetization between them to the proton relaxation behavior was originally proposed by Swift and Connik. this the first evidence to account for anisotropy in chitosan hydrogels at neutral and alkaline pH. T1 and T2 Relaxation at Varying pH In heterogeneous polymer systems the observed proton NMR relaxation (T1 and T2) behavior is determined both by the intrinsic relaxation of each of the (motionally inequivalent) proton populations and by the effects of magnetization transfer that may occur between them. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co. . Hence. as can be appreciate on Table 2. Weinheim 893 .e. This approach requires a detailed study of the temperature dependence of T1 and T2. while the other is due to the contribution of ionized –O– groups themselves that. hence. www. Chem. the greater charge density of the polymer network will result in a more expanded network enabling rapid diffusion of solvent species. The existence of lyotropic liquid crystalline mesophases in chitosan and chitosan derivatives concentrated acidic solutions has been reported. Because of this delay. One is that as the –OH groups of the polysaccharide are gradually ionized.e.mcp-journal.and Temperature-Sensitive Chitosan Hydrogels . the gel network is expected to achieve full neutrality (i.e. is accompanied by a progressive elevation in T1 relaxation time from 1 580 to 1 800 ms.2[33]). It is interesting to point out that the variation of T2 values seems to be closely dependent on the equilibrium amount of water in the hydrogels given by the degree of swelling. the signal from the polymer protons is effectively dephased and does not make a measurable contribution to the observed T1. By contrast. to the best of our knowledge. One is given by motions of protons associated with rigid parts of the network (i. Whereas molecular motions of protons in more flexible parts of the gel network (i.0.g. –CH2 and C1–C5. at pH ¼ 8.

J. showing the ROIs considered in the calculations. Makromol. [12] J. [4] M. 2009. Polym. Peppas. 429. M. ADC MRI determinations have revealed that there is anisotropy in these hydrogels and water diffuses more rapidly along the radial direction than the axial one. Kandani. A. Bushman. 887–895 ß 2011 WILEY-VCH Verlag GmbH & Co. 2001. T1 and T2 parametric coronal maps of chitosan hydrogels at the three studied pH values. Mayer. Pharm. At such pH and at low temperatures ( 2 8C) the gels are greatly swollen. Biomaterials 2005. F. Vila-Jato. F. 951. M. 2000. KGaA. K. Biomacromolecules 2007. 2010. J. J. thus this approach was not applied to our data. 2804. Y. R. 26. 5983. 76. [2] J. [10] R. Biomaterials 2005. Rinaudo. Roberts. Figure 5 shows representative diffusion. These systems respond to changes in their swelling state with stepwise changes in temperature within a narrow range of pH and this response is more pronounced in the vicinity of pH  7. Nakamatsu.mcp-journal. 2010. Chem. 2001. Taylor. A. I. Ferna´ndez-Valle. Chaput. when the wound is open. 63. applications.´ . N. 452. 1997. Int. Biopharm. Gurny. F. Muzzarelli. 2011. M. Phys.0. Shin-Shing. Vachoud. SB99-B98260006). MRI has been proved as a powerful and non-invasive technique to probe the state of water in chitosan hydrogels at varying pH. Eur. T1 and T2 shown in Table 1 and 2. M. Indeed. and pH  7. Sittinger. Received: May 28. A www. Polym. 43450) are gratefully acknowledged. Troncoso. The thermosensitive and pH-sensitive properties of these hydrogels can be utilized in the development of innovative materials for biotechnological and biomedical 894 Keywords: chitosan. E. hydrogels. stimuli-sensitive [1] G. 212. Published online: February 25. 46. Chem. MAT2000-0037-P403). Sci. A. [5] P. as the net amount of water decreases it is expected that rotational and translational motion of the water molecules will be smaller. A. DOI: 10. M. David. O. Chem. 21. C. [6] L. Macromol. Min-Lin. Goycoolea. Wang. magnetic resonance imaging (MRI). 190. by virtue of the protonation of –NH2 and ionization of –OH groups in chitosan. Gels Networks 1998. 28. J. N. Berger. C. It was also demonstrated that the state of water is clearly governed by the pH that controls the ionization state of the gel. Martinez. [13] I. S. swelling. Hsing-Wen. M. Macromol. Fwu-Long. The results are consistent with the notion that the average mobility of water molecules (bound to increase with swelling degree) is the dominant factor in the chemical exchange processes. R. M. Carbohyd. It must be noticed that the intensity of the images in each map corresponds with the calculated values for D. Heras F. [3] W. N. Part A: Polym. 6. Risbud. Calvo. Adekogbe.com .201000301 Conclusion The results of this work confirm those of previous studies that have addressed the behavior of chitosan hydrogels obtained from alkali chitin and found to exhibit a phase transition temperature centered at 20 8C. 2011. 7241. Int. Domard.6 as the wound is healed. including criobiocatalysis and bioremediation as well as in programmed drug delivery. M. Weinheim www. T1 (b) and T2 (c) for chitosan hydrogels at 2 8C and varying pH (as shown in labels). P. M. [11] A. 1996. S. Argu ¨ elles-Monal.. Di Martino. 7. D. Peniche. The treatment of open wounds for example. Desbrieres. I. M.MaterialsViews.6. [9] S. whereas as the temperature increases to 25 8C and beyond. L. the CYTED (Project XIV and Network IVG) and CONACYT (Project No. Polym. 76. At 37 8C no pHdependence in swelling was observed. A. Biol. Boccaccini. C. A. Alonso. 3345. 125. Ladet. C. Reist. J. [8] J. Zydowicz. MRI coronal parametric maps of D (a). J. Appl. Polym. V. the Ministry of Science and Technology of Spain (Project No. 167. 38. [7] A. Aranaz. 26. will benefit from a dressing that utilizes a chitosan hydrogel that changes can experience a change in its swelling and diffusion properties between pH 8. Macromol. Revised: December 24. 39. A. 1934. D. Sci. Chenite.1002/macp.[38] Acknowledgements: Grants from the Ministry of Education and Culture of Spain (Project No. HigueraCiapara. 19. It can be appreciated that all images display fair radial signal homogeneity across their diameter. Nature 2008. G. O. Ghanem. Torres. A.de Figure 5. 93. the gels are collapsed. Domard. Carbohyd. Biol. J. L. Remun ˜ a´n-Lo´pez. Polym. 57. J. Goycoolea. 2004. Felt. E. 1989. a precise control of the sample temperature during the measurements.

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