Covalent Co

ARTICLE pubs.acs.org/Biomac

In Situ Forming Chitosan Hydrogels Prepared via Ionic/Covalent Co-Cross-Linking M. Jose Moura,† H. Faneca,‡ M. Pedroso Lima,‡ M. Helena Gil,§ and M. Margarida Figueiredo*,§ †

Departamento de Engenharia Química e Biologica, Instituto Superior de Engenharia de Coimbra, R. Pedro Nunes, 3030-199 Coimbra, Portugal ‡ Centro de Neurociências e Biologia Celular and Departamento de Ciências da Vida, Universidade de Coimbra, 3004-517 Coimbra, Portugal § Centro de Investigac-~ao em Engenharia dos Processos Químicos e Produtos da Floresta and Departamento de Engenharia Química, Universidade de Coimbra, P olo II, Pinhal de Marrocos, 3030-290 Coimbra, Portugal ABSTRACT: In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerolphosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerolphosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.

’ INTRODUCTION Injectable, in situ forming hydrogels are of great interest as components of drug delivery systems, cell encapsulation vehicles, and scaffolds for tissue engineering.13 In their use, such hydrogels offer several advantages over conventional solid implants. These advantages include their low cost and easy handling, the reality of reduced scarring and healing periods, less pain to the patient, minimized surgery time, and good conformation to irregular defects.48 In general, these formulations have been developed as microcapsules or microspheres. However, in microparticulate systems, their efficiency in the drug absorption of cytotoxic molecules is limited, and high loading capacities are unattainable.6 Such problems have oriented the scientific community toward the development of injectable, in situ gelling formulations, such as those provided by chitosan-based combinations. Although several chitosan hydrogels have been investigated, only a few of these possess in situ gelling properties.6 Chitosan hydrogels have been prepared by the physical crosslinking or the chemical cross-linking of polymer chains.912 r 2011 American Chemical Society

With respect to the former approach, the addition of specific basic salts, such as glycerolphosphate complex, results in an increase in the solution pH due to the neutralizing effect of the phosphate groups. This enables the transformation of the original solution into a temperature-controlled, pH-dependent matrix. Chemical cross-linking, leading to the creation of hydrogel networks that possess improved mechanical properties and chemical stability,10,11 can be achieved using either synthetic agents or natural-based agents. Glutaraldehyde has been extensively employed as a chemical cross-linker agent.13 However, it has been shown that glutaraldehyde exhibits cell cytotoxicity. The tendency has been to replace glutaraldehyde with naturally occurring cross-linking agents, such as genipin.14 Physically cross-linked chitosan gels are fragile, have low mechanical Received: May 30, 2011 Revised: July 18, 2011 Published: July 20, 2011 3275

dx.doi.org/10.1021/bm200731x | Biomacromolecules 2011, 12, 3275–3284

Biomacromolecules integrity, and exhibit a high degradation rate, especially in acidic environments or in the presence of lysozyme.11,15 The purpose this Article was to investigate the influence of the addition of different amounts of genipin to a chitosan solution that was previously neutralized with glycerolphosphate on the potential of these formulations to act as injectable, in situ forming gels. With such formulations, it could be possible to overcome the pH barrier of the chitosan solution, to take advantage of its thermosensitive character, and to enhance the matrix crosslinking degree under mild conditions of pH and temperature. Although the interactions of chitosan with glycerolphosphate (ionic cross-linking) and of chitosan with genipin (covalent cross-linking) have been described in the literature,1,9,14,1618 the combination of ionic cross-linking mechanisms and covalent cross-linking mechanisms has been seldom studied.19 In previous work,20,21 the present authors have shown that formulations that were prepared under physiological conditions (pH 7 and 37 °C), by simultaneously cross-linking chitosan with a glycerolphosphate disodium salt combination and different loadings of genipin (up to 0.20%), not only maintained the thermosensitivity of the chitosan-based matrix but also produced relatively strong elastic gels. Moreover, it has been shown that different cross-linker concentrations can be employed to modulate the gelling ability of chitosan solutions, with there being a strong impact on the gelation time. The emphasis of the present report is placed on the evaluation of the effects of using different amounts of genipin (including 0%) on the extent of cross-linking, the swelling behavior, the morphology, the degradability, and the cytotoxicity of the chitosan/glycerolphosphate hydrogels. In attempting to differentiate between the physical cross-linking effects and the chemical cross-linking effects, additional tests were performed using chitosan that was cross-linked with genipin only, at the same loadings as those used in the neutralized formulations.

’ EXPERIMENTAL SECTION Materials. The chitosan used in the experiments, (molecular weight ∼2 105 Daltons and a degree of deacetylation of 87%, calculated from the carbon/nitrogen ratio by elemental analysis), was purchased from Sigma-Aldrich, in a powder form. The hydrated glycerolphosphate disodium salt (C3H7Na2O6P 3 xH2O; FW = 218.05), used to adjust the pH of the chitosan solutions, was also obtained from Sigma-Aldrich. Genipin (crystallike powders, reagent grade) was supplied by Challenge Bioproducts, Taiwan. Phosphate-buffered saline (PBS, pH 7.4), obtained from Sigma-Aldrich, was prepared by dissolving one tablet in 200 cm3 of distilled water. Lysozyme (crystalline) from egg white (∼40 000 units/mg) was purchased from B. D. H. Laboratory Chemicals Division, England. Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG), fetal bovine serum (FBS), trypsin solution (0.25%), and rezasurin (Alamar Blue assay) were obtained from the Sigma Chemical (St. Louis, MO). All of the other reagents and solvents that were used in this work were of the highest purity commercially available. Preparation of Chitosan Hydrogels. The genipin-cross-linked chitosan/glycerolphosphate hydrogels (C/GP/GE) and the chitosan/ glycerolphosphate hydrogels (C/GP) were prepared at neutral pH and at body temperature, according to Moura et al.20 In brief, a solution containing 2 g of chitosan in 100 cm3 of volume was prepared by dissolving the chitosan powder (C) in distilled water that contained 0.5% (by volume) acetic acid at room temperature. The pH was adjusted to 7 by the addition of the required amount of glycerolphosphate disodium salt (GP), previously dissolved in distilled water. The final

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solution contained 1.5 g of chitosan in 100 cm3 of solution and 5.5 g of GP in 100 g of solution. Afterward, genipin powder (GE) was dissolved in the chitosan solution, which was finally placed in a polysiloxane mold to produce cylindrical samples (diameter 14 mm). Different amounts of genipin were used: 0.05, 0.10, 0.15, and 0.20% (mass of genipin/mass of total solution), the corresponding samples being designated as C/GP/ GE5, C/GP/GE10, C/GP/GE15, and C/GP/GE20. As mentioned above, to separate the effects of ionic interaction (physical) and the effects of chemical cross-linking, chitosan hydrogels were prepared without genipin (C/GP) and with genipin (but without the addition of glycerolphosphate), subsequently denoted as C/GE5, C/GE10, C/GE15, and C/GE20, respectively, corresponding to 0.05, 0.10, 0.15, and 0.20% of genipin (by weight). The hydrogels produced were subsequently cured at 37 °C, for 24 h, and then further characterized, as mentioned above. Fourier Transform Infrared Spectroscopy. Fourier transform infrared (FTIR) spectroscopy was used to investigate the contribution of ionic cross-linking and of chemical cross-linking on the produced chitosan-based hydrogels. The hydrogels were lyophilized for 3 days (Snijders Scientific type 2040, Tilburg, Holland), subsequently ground to powder, and then mixed with KBr (0.8 mg sample/80 mg KBr). The mixed samples were pressed to a disk format (7 mm of diameter). FTIR spectra were taken with a resolution of 4 cm1 (32 scans), on a Jasco, FTIR-4200 spectrometer (Tokyo, Japan). FTIR spectra of original chitosan and glycerolphosphate, as KBr discs, were also recorded. Ninhydrin Assays. This assay enabled the determination of the percentage of free amino groups of the chitosan-based hydrogels, after the cross-linking reaction.18,22 About 2 mg of lyophilized sample was heated to 100 °C in a water bath, with 1 cm3 of the ninhydrin solution, for 20 min. Subsequently, the solution was cooled to room temperature and diluted with 5 cm3 of 50% isopropanol. The optical absorbance of each solution was determined at 570 nm using a MAPADA, UV-1800 spectrophotometer (MAPADA Shanghai, China). The same procedure was followed for assessing the chitosan powder (used as control). The optical absorbance of the solution is proportional to the number of free amino groups in the test sample, determined after heating the sample with ninhydrin. The absolute values were computed from a standard curve of the glycine concentration versus the absorbance.18,22 The degree of cross-linking of the gel (in percentage) was then calculated according to eq 1 degree of cross-linking ¼

ðNHN reactive amineÞfresh ðNHN reactive amineÞfixed ðNHN reactive amineÞfresh

100

ð1Þ Here the term “NHN reactive amine” represents the mole fraction of free NH2 groups, the designation “fresh” corresponds to the original chitosan sample (non-cross-linked), and “fixed” corresponds to the cross-linked samples. At least four samples of each hydrogel system were tested (either C/GP/GE or C/GE) in addition to the tests carried out on the chitosan powder (C). Morphological Characterization. The microstructure of the chitosan/glycerolphosphate hydrogels that had been cross-linked with different loads of genipin was examined using a scanning electron microscope (SEM). For this purpose, the hydrogel samples were frozen at 20 °C and further freeze-dried in a lyophilizer (Snijders Scientific type 2040, Tilburg, Holland) under vacuum (0.50 mbar) at 50 °C for at least 3 days, until all of the solvent had sublimed. The dehydrated samples were cross-sectioned and placed on double-sided tapes, sputtercoated with gold, and observed by SEM (model JSM-5310, JEOL, Tokyo, Japan). Additional measurements were performed on the samples under wet conditions, following the protocols for ESEM imaging, as described in the literature,23 using a high-resolution environmental scanning electron microscopy unit (ESEM, FEI Quanta 3276

dx.doi.org/10.1021/bm200731x |Biomacromolecules 2011, 12, 3275–3284

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Figure 1. FTIR spectra of (A) original chitosan powder (C), the glycerolphosphate disodium salt complex (GP), the chitosan/glycerolphosphate hydrogel (C/GP), the chitosan hydrogel system that was cross-linked with 0.20% genipin without the addition of glycerolphosphate (C/GE20), the chitosan/glycerolphosphate hydrogel system that was cross-linked with 0.20% genipin (C/GP/GE20). (B) Section of part A showing an enlargement of a section in the region 1300800 cm1. 400 FEG) equipped with an energy dispersive X-ray spectrometer (EDAX Genesis X4M). The porosity and the pore size distribution of the freeze-dried hydrogels were further quantified by mercury intrusion porosimetry (MIP) using the AutoPore IV 9500, from Micromeritics, over a range of pore sizes between 400 and 0.006 μm. Swelling Measurements. Swelling tests were performed on the chitosan/glycerolphosphate hydrogels (∼700 μL) with their different chemical cross-linker loads, prepared at neutral pH and the body temperature (37 °C). After curing, the gels were removed from the mold and subsequently incubated in PBS at 37 °C for 24 h to reach their equilibrium swelling state. The excess of incubating medium was removed, and the surface water on the hydrogels was blotted gently

with a filter paper. The gels were subsequently weighed (We). Afterward, the hydrogels were frozen at 20 °C and then transferred to a lyophilizer (Snijders Scientific type 2040, Tilburg, Holland), where they were freeze-dried at 50 °C and 0.50 mbar for 3 days and then weighed (Wd). All of the experiments were performed in triplicate. The equilibrium water content (EWC) was calculated using eq 2 EWC ð%Þ ¼

We Wd 100 We

ð2Þ

In Vitro Enzymatic Degradation. The rate of degradation of the chitosan samples was determined gravimetrically for various hydrogel 3277


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Figure 2. Effect of genipin load on the number of free —NH2 groups (in terms of the glycine concentration) in chitosan hydrogels that were previously neutralized with the glycerolphosphate complex. Sample C/GP does not contain genipin. The dashed line corresponds to the original chitosan powder. Asterisks (*p < 0.05, **p < 0.01, ***p < 0.001) indicate values that differ significantly from those obtained with the C/GP hydrogel. Statistical analysis shows that most of the conditions (different genipin loads) are significantly different (p < 0.05 for C/GP/ GE5 when compared with C/GP/GE10 and for C/GP/GE10 when compared with C/GP/GE20; p < 0.01 for C/GP/GE5 when compared with C/GP/GE15; p < 0.001 for C/GP/GE5 when compared with C/GP/GE20), except for C/GP/GE10 when compared with C/GP/ GE15 and for C/GP/GE15 when compared with C/GP/GE20. types (C/GP, C/GP/GE5, C/GP/GE10, C/GP/GE15, and C/GP/ GE20) cured for only 2 h at 37 °C. This short curing time was selected so that the material could be analyzed under conditions that were as close as possible to those that would be applied in in vivo situations, ensuring that the samples were in a solid form at the beginning of the tests. The mass of the hydrogels (∼0.4 g), after reaching equilibrium in PBS at 37 °C for 24 h was monitored as a function of the incubation time (up to 28 days) in 10 cm3 of PBS at 37 °C, with gentle agitation in an orbital shaker. To mimic the physiological conditions of the in vivo degradation, 1.5 μg of lysozyme/cm3 of solution was added. This concentration of lysozyme was chosen to correspond to that in human serum.24,25 The lysozyme solution was refreshed every 2 days to simulate continuous enzyme activity. At specified time intervals, hydrogels were removed from the medium, blotted gently with a filter paper to remove the surface water, and finally weighed. The extent of in vitro degradation was expressed as percentage of weight loss using eq 3 Wi Wt 100 weight loss ð%Þ ¼ Wi

ð3Þ

Here Wi and Wt, respectively, denote the initial weight and the final weight of the samples. Additionally, to establish whether there was any impact on enzymatic degradation caused by dissolution or dehydration phenomena, samples were submitted to degradation tests under the same conditions as those outlined above, without the addition of the lysozyme solution. Three samples of each type of hydrogel were evaluated. Cytotoxicity Studies. Cell Culture. TSA cells (BALB/c female mouse mammary adenocarcinoma cell line) were maintained at 37 °C, under 5% CO2, in DMEM-HG supplemented with 10% (v/v) heatinactivated FBS, penicillin (100 U/cm3), and streptomycin (100 μg/cm3). TSA cells grow in a monolayer and were detached by treatment with a trypsin solution (0.25%). We seeded 0.5 105 TSA cells in 1 cm3 of medium in 48-well culture plates, 24 h before incubation, to obtain 5070% confluence. Cell Viability Assay. For these assays, the gels were prepared as described above but using materials that were previously sterilized. A sterile chitosan solution was obtained by autoclaving (121 °C, 15 min).

The glycerolphosphate disodium salt was dissolved in distilled water and sterilized by filtration through a 0.20 μm pore size disk filter. The genipin powder was sterilized by exposure to ultraviolet radiation for 2 h. All of the hydrogel formulations were prepared in a laminar-flow hood under aseptic conditions. The cytotoxicity of the gels was evaluated by an extraction test according to ISO 10993-5 Standard.26 The gels were immersed in a culture medium (DMEM-HG) at an extraction ratio of 1 cm3 per 1.25 cm2 of gel surface area1 and incubated in a humidified atmosphere with 5% carbon dioxide, for 24 h, at 37 °C. TSA cells, seeded in 48-well culture plates, were incubated with extraction fluid for 48 h, and the cell viability was assessed by the use of a modified Alamar Blue assay.27 In brief, following incubation of the cells for 48 h with the extraction medium, the medium of each well was replaced with 0.3 cm3 of DMEMHG, containing 10% (v/v) of Alamar Blue (0.1 mg/cm3 in PBS). After 1 h of incubation at 37 °C, 200 μL of the supernatant was collected from each well and transferred to 96-well plates. The absorbance at 570 nm and at 600 nm was measured spectroscopically (SPECTRAmax PLUS 384, Molecular Devices, Union City, CA). The cell viability was calculated as a percentage of the control cells (cells not treated with the extraction medium) according to eq 4 cell viability ¼

ðabsorbance570nm absorbance600nm Þtreated cells 100 ðabsorbance570nm absorbance600nm Þcontrol cells

ð4Þ In Vivo Hydrogel Formation. To establish whether the chitosan hydrogels would rapidly form a gel at the injection site, we administered a liquid chitosan/glycerolphosphate formulation containing 0.10% of genipin (C/GP/GE10), with pH value of 7.08, by dorsal subcutaneous injection into an adult male Wistar rat (∼ 300 g), following the European Community guidelines. A sterile formulation was produced in exactly the same way as that developed for the cell viability assays. Thus, a rat was anesthetized by intraperitoneal injection of ketamine and chlorpromazine, and made sterile for three dorsal injections. Each injection (0.3 mL in volume) was performed through a syringe that was equipped with a gauge 21G needle. Gel formation at the injection sites was verified visually. The volume of hydrogel in the rat was measured using a Helios Vernier caliper every 48 h after injection and was calculated by following eq 528 volume ðmm3 Þ ¼ length width2 1=2

ð5Þ

After 7 days, the rat was sacrificed and the chitosan-based hydrogels were localized. Statistical Analysis. Statistical analyses of the data were performed using the GraphPad Prism software (version 5.0). The statistical significance of the differences between data was evaluated by a oneway ANOVA using the Tukey test. A value of p < 0.05 was considered to be significant. All of the data are presented as the mean ( standard deviation.

’ RESULTS AND DISCUSSION Fourier Transform Infrared Spectroscopy. Figure 1 shows the FTIR spectra of the chitosan-based hydrogels together with those of the chitosan powder and of the glycerolphosphate salt. The chitosan spectrum (C), besides having the band in the region of 35003200 cm1, corresponding to the amino group that is masked by the broad absorption band from the OH group, exhibits an absorption band with maximum at 2868 cm1, representing the CH2 and CH3 aliphatic groups.29 The peak at 1645 cm1 is assigned to the CdO stretch of the amide bond, and the absorption band at 1586 cm1 corresponds to the primary amine groups.14,18 As for the glycerolphosphate complex (GP), 3278


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the corresponding spectrum shows an intense absorption within 1050 to 1150 cm1 that is due to the PO and PdO vibrations. The peak at 970 cm1 is normally assigned to the POH group.30 The multiple peaks in the spectrum of the genipin (not included here to avoid any image overload of Figure 1 but available in the literature14,18) that centered around 1700 cm1 and are attributed to the CdO stretching of genipin molecule are probably more relevant to this study. Examination of the spectrum that is associated with chitosan that had been neutralized with the glycerolphosphate solution (C/GP) shows that although bands that are characteristic of the chitosan and of the glycerolphosphate complex can be detected in the spectrum of C/GP, this does not imply the addition of the peaks corresponding to the constituents. This point is more evident in Figure 1B, which depicts the enlargement of the footprint region (1300800 cm1). The observed differences are probably the result of ionic interactions between the positively charged amino groups of the chitosan and the negatively charged phosphate groups of the GP. Similarly, the FTIR spectrum of the genipin-cross-linked chitosan hydrogels, without the glycerolphosphate (C/GE20), exhibits some differences that are related to the spectra of its constituents (Figure 1A). It was observed that the absorbance at 1583 cm1, corresponding to primary amine groups of the Table 1. Influence of Genipin Loading on the Gelation Time, tg, and on the Elastic Modulus, G0 , of Chitosan Hydrogels That Had Been Previously Neutralized with the GlycerolPhosphate Complex20 hydrogel

gelation time, tg (s)

elastic modulus, G0 (Pa)

C/GP

519

3

C/GP/GE5

312

65

C/GP/GE10

187

196

C/GP/GE15

113

1030

C/GP/GE20

3120

chitosan, decreased after the genipin treatment had been applied because of the chemical change of these functional groups during the chemical cross-linking reaction. Furthermore, the change in the maximum from 1645 to 1676 cm1 and the apparent increase in the intensity of this peak, together with the drastic reduction of the intensity of CdO stretching from genipin at ∼1700 cm1 indicates the formation of the secondary amide group as a result of the reaction between the amino groups of the chitosan and ester groups of the genipin.14 In the FTIR spectrum of the co-cross-linked hydrogels (C/ GP/GE20), the occurrence of chemical cross-linking is, once again, apparent. Finally, it is noteworthy that the characteristic peak for genipin vanished completely. As illustrated in Figure 1B, the peaks in the region corresponding to the phosphate functionalities (1300800 cm1) appear to be much less resolved compared with the resolution obtained in the spectrum of the C/GP, suggesting that the ionic interactions are more attenuated in the co-cross-linked network. Ninhydrin Assays. To determine the extent of the various types of cross-linking reactions, we performed ninhydrin assays on all of the samples. As mentioned in the Experimental Section, this assay quantifies the amount of free amino groups in the test sample, expressed in terms of the glycine concentration. To guarantee the complete occurrence of the crosslinking reactions, we cured the hydrogels used in these tests for 24 h. Figure 2 shows the results that were obtained for the chitosanbased formulations that had been neutralized with the glycerol phosphate salt complex for the different percentages of genipin (including 0%). In Figure 2, the dashed line represents the amount (micromoles of glycine per milligram of sample) corresponding to the non-cross-linked chitosan (2.47 μmol/mg). These data reveal that the sample corresponding to the hydrogel that was ionically cross-linked, that is, without genipin (C/GP), contained the least amount of free amino groups (∼0.25 μmol/ mg, equivalent to a percentage of ∼10%), probably as a result of the great extent of ionic interaction. In fact, as described in the

Figure 3. Schematic representation of chitosan-based hydrogel formation under the specified physiological conditions via (A) ionic cross-linking and (B) ionic cross-linking and covalent cross-linking. 3279


Biomacromolecules literature,30 phosphate ions can ionically interact with the NH3+ bind sites in chitosan. However, ionic interactions can also occur without necessarily involving ionic cross-linking of polymeric chains. The addition of genipin drastically increases the quantity of free amino groups, which, in turn, decreases as the genipin concentration increases. Therefore, the addition of the chemical cross-linker tends to disturb the ionic cross-linking mechanism, suggesting that the chemical cross-linking process is the dominant process. This is to be expected because the loss of mobility of the chitosan chains due to covalent interactions limits the possibility of optimal ionic interactions taking place. This observation is in accord with the results obtained from the FTIR analyses. Therefore, the peaks corresponding to the ionic interactions are better resolved in the C/GP spectrum than in the spectrum of the C/GP/GE20 system. The results from a previous study20 (Table 1) have shown that in terms of mechanical properties, the addition of genipin to neutralized chitosan solutions has a strong impact on the reduction of the gelation time and on the enhancement of the network elasticity. The results from the current study clearly indicate that the improvement in the stability of the 3-D crosslinked networks is a consequence of the stronger interactions that occur through covalent cross-linking than through ionic cross-linking. On the basis of these observations, Figure 3 schematically illustrates a possible cross-linking mechanism for the ionic

Figure 4. Cross-linking degree (calculated from eq 1) for genipincross-linked chitosan hydrogels with and without the addition of the glycerolphosphate salt.

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reactions and for the case where ionic cross-linking and covalent cross-linking occur simultaneously. To investigate further the effects of physical cross-linking and of chemical cross-linking, we prepared a set of additional samples, this time without previous phosphate neutralization having been applied, for the same genipin concentrations. In these formulations, the prevailing pH was 5.4 (rather than 7). Similar ninhydrin assays were also conducted on these samples, leading to the results depicted in Figure 4, expressed in terms of the percentage of cross-linking degree (eq 1). Figure 4 suggests that the degree of cross-linking is always less in the chemically cross-linked samples than in those samples that were prepared by co-cross-linking. This observation can be related not only to the effects of both the ionic cross-linking and the chemical cross-linking but also to the pH effect. As mentioned above, the pH of the solely chemical cross-linked samples is less than that of the formulations that were neutralized with the glycerol phosphate salt complex. As reported in the literature,18,19 the cross-linking reaction that is associated with the cross-linking of chitosan (using tripolyphosphate and genipin) is highly dependent on the solution pH, achieving the greatest extent of crosslinking under neutral conditions. Regardless of the absolute values obtained for the samples that had been processed with and without the use of the glycerol phosphate complex, it is evident, from the results that were

Figure 5. Effect of the genipin concentration on the equilibrium water content of chitosan hydrogels that had been previously neutralized with the glycerolphosphate complex (including 0% genipin (C/GP)). Asterisks (***p < 0.001) indicate values that differ significantly from those obtained with the C/GP hydrogel. Statistical analysis also shows that all conditions (different genipin loads) are significantly different (p < 0.001), except the C/GP/GE15 when compared with C/GP/GE20 hydrogel.

Figure 6. SEM photomicrographs of chitosan matrices: (A) chitosan neutralized with glycerolphosphate hydrogels (C/GP) and (B) chitosan neutralized with glycerolphosphate hydrogels with 0.10% genipin (C/GP/GE10). The inset pictures present a higher magnification of the pore walls. 3280


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Figure 7. Images of the chitosan matrices, illustrated in Figure 6, as evaluated using environmental SEM (under wet conditions).

Figure 8. Differential pore size distributions that were obtained for the samples that are illustrated in Figure 6 (chitosan neutralized with glycerolphosphate hydrogel (C/GP) and chitosan neutralized with glycerolphosphate hydrogel with 0.10% genipin (C/GP/GE10)) measured by mercury intrusion porosimetry. The values of the total porosity of these samples are 97 (C/GP) and 93% (C/GP/GE10), respectively.

obtained, that the genipin load can be used to modulate the extent of cross-linking of the networks and consequently the mechanical properties of the networks (Table 1). It should be stressed that although the studied formulations that possess pure covalent bonds exhibit apparently lesser extents of cross-linking, they are also of limited use to the production of in situ hydrogels because of the acidic character of the system (unsuited to the delivery of bioactive materials). For all of these reasons, such samples were not subjected to further characterization. Swelling Measurements. On the basis of the results presented in Figure 5, it is clear that the greatest values of EWC correspond to the chitosan hydrogel that does not contain genipin (C/GP). This is apparently in contradiction with the results given in Figure 2 that revealed that this sample contained the least amount of free amino groups. However, this difference can be explained by the interpretation that the ionic cross-linking is not strong enough to reduce the mobility of the polymer chains and, consequently, to modify the expansion of the gel that is in contact with the solvent. This observation is compatible with the

Figure 9. Effect of genipin concentration on the degradation profiles of the chitosan hydrogels that had been previously neutralized with the glycerolphosphate in PBS, with 1.5 μg of lysozyme/cm3, at 37 °C. Sample C/GP does not contain genipin. Triplicates of each hydrogel were analyzed, and each datum point represents the mean value ( standard deviation.

weak mechanical properties that were exhibited by this gel (given longest gelation time and least elastic modulus, Table 1). However, and in agreement with the findings from the ninhydrin assays, as the genipin concentration increases, the swelling properties of the corresponding hydrogels consistently decrease, indicating the reduction in the number of available hydrophilic groups and the increase in the extent of the crosslinking. Furthermore, the reduction in the sample swelling, corresponding to the greater genipin loadings (0.15 and 0.20%), tends to be attenuated. (The swelling of C/GP/GE15 and C/GP/GE20 hydrogels is not statistically different, p > 0.05.) Nonetheless, all of the hydrogels exhibit the remarkably high EWC (>90%), that is commonly found for these types of gels.14,18,31 Additional characterization procedures were applied to these samples to evaluate some of their properties. These procedures include morphological assessments, degradation studies, and cytotoxicity evaluations. 3281


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Figure 11. Effect of the genipin loads (including 0%) on cell viability. The TSA cells were incubated for 48 h in the different extraction media; then, the cell viability was measured by the Alamar Blue assay procedure. The data are expressed as the percentage of the untreated control cells (mean ( standard deviation obtained from n = 6) and are representative of three independent experiments. Statistical analysis indicates that the cell viability values are not significantly different from those obtained with the C/GP hydrogel.

Figure 10. Comparison of the weight loss profiles of selected chitosan hydrogels in PBS without lysozyme (O) and with lysozyme (b) at 37 °C. Triplicates of each hydrogel system were analyzed, and each datum point represents the mean value ( standard deviation.

Morphological Characterization. In the present study, the internal morphology of the hydrogels was analyzed by “conventional” SEM, by environmental SEM, and by MIP. Figure 6 presents SEM micrographs of the internal structure of a neutralized chitosan hydrogel without genipin (A) and with 0.10% of genipin (B), imaged after water removal by freezedrying. An open network structure is shown that appears to be highly porous with the interconnected macrodomains. This morphology comprises the polymer and pores as features not only of these samples but also of all of the samples that were produced. These pores possess similar shapes, with a polygonal cross section. However, those corresponding to the matrices that were cross-linked with genipin are much larger (a few hundred micrometers for the samples that contained genipin, while being only a few tens of micrometers for those matrices that were ionically cross-linked) and are less uniform in size. Moreover, the pore size and morphology of the various hydrogels that were cross-linked with genipin were quite similar, regardless of the

genipin loading that was used (data not shown). The pore walls of the samples containing genipin are also considerably thicker than those of the corresponding ionically cross-linked sample. On the understanding that the freeze-drying conditions might affect the final microstructure of the samples, an attempt was made to obviate the need for sample drying by using the ESEM technique. This technique allows high-resolution imaging to be undertaken when the samples is contained at a high relative humidity. The corresponding micrographs are presented in Figure 7. These images confirm that the samples with genipin exhibit pore sizes that are substantially larger than those prepared without the use of the chemical cross-linker. Additionally, the images also suggest that the pore sizes of the dried samples are close to those of the hydrogels in their wet form. As expected, the changes observed in the SEM images, as shown in Figure 6 for the freeze-dried samples, are substantiated by the pore size distributions that were determined by MIP, as carried out on these samples, illustrated in Figure 8. In fact, a narrow peak, corresponding to the curve relating to the ionically cross-linked chitosan hydrogels has a mode at ∼60 μm, whereas that of the cross-linked matrix exhibits a broader size distribution, with a mode at ∼200 μm. It should be noted that these curves may not fully describe the pore size distribution of the samples because MIP is not able to measure pores that are greater than ∼400 μm. In fact, Figure 8 suggests that in the case of the C/GP sample no pores larger than the detection limit should be expected. However, in the case of the genipin-cross-linked hydrogel (C/GP/GE10), the size distribution curve is clearly truncated at the upper detection limit. This is why the porosity values measured by MIP (97% for the sample C/GP versus 93% for the sample C/GP/GE10, as indicated in the Figure caption), especially for the sample with genipin, should be regarded with caution, being probably underestimated. Nonetheless, it is noteworthy that all of the hydrogels are highly porous, exhibiting porosities >90%. These findings confirm the supposition that chitosan/glycerolphosphate/genipin gels should be highly promising materials with respect to their employment as scaffold components, allowing nutrients to diffuse easily inside the matrix. 3282


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Cytotoxicity Studies. The results presented in Figure 11 reveal that the viability of the TSA cells was not significantly affected upon incubation with each of the gels extraction media that were used, (either without genipin or with different genipin amounts). Moreover, the results also show that the use of genipin up to 0.20% did not result in any decrease in the cell viability. In Vivo Hydrogel Formation. A formulation that contained ∼0.10% of genipin (C/GP/GE10) was further tested by injecting it into a rat subcutaneously (Figure 12A). Besides wishing to monitor the injectability of the composition, another objective of this in vivo test was to ensure that the gel was rapidly formed and was not delocalized throughout time. Figure 12B shows that after 7 days, the gel samples were located in exactly the same position as that at which the initial deposition had taken place and did not leak to the surrounding tissues (Figure 12C). Furthermore, the implant volume (∼2000 mm3) was approximately the same throughout the time. In addition, during this period, the rat did not exhibit any side effects (such as pus or inflammation) and was in perfectly good health.

Figure 12. Image of the subcutaneous gel implants after dorsal injection in an adult Wistar rat of a chitosan formulation (C/GP/GE10): (A) before and (B) after 7 days of administration. (C) Gel localized within the subdermal fibrous membranes.

In Vitro Enzymatic Degradation. In the present work, the degradability (as indicated by the rate of any degradation reaction) of chitosan/glycerolphosphate hydrogels that have been cross-linked with different, known amounts of genipin was studied by monitoring the weight loss of samples, with time, in a PBS environment, at 37 °C, for 28 days. In an initial approach, to mimic the in vivo degradation performance, lysozyme was added to PBS solution. Figure 9 illustrates the effect of the genipin loading (including 0%) on the gel weight loss as a function of time. As expected, the faster weight loss rate was found for the chitosan/glycerolphosphate hydrogel without genipin (C/GP), which completely disintegrated after 9 days. As for the co-cross-linked gels, these gave an exponentially based degradation rate that was considerably lower than that of the purely ionically cross-linked chitosan, with the greatest rates occurring up to 10 days. After 28 days, the weight losses were ∼80 and 40%, respectively, for the least genipin loading and for the greatest genipin loading. To establish the importance of the contribution of the enzymatic degradation and of simple dissolution (and/or dehydration) to the total weight loss, additional tests were performed using samples that had been immersed in PBS without lysozyme. The results from this comparative study are presented in Figure 10 for selected genipin loadings (C/GP, C/GP/GE5, and C/GP/GE20). The degradation dynamics of the samples that were immersed in PBS, either with or without the lysozyme, clearly highlights the relevance of enzymatic degradation, especially for those samples with the lesser extents of cross-linking.

’ CONCLUSIONS This work demonstrates that it is possible to prepare in situ gelling chitosan hydrogels that can be used under physiological conditions, providing enhanced network properties via ionic cross-linking and via covalent co-cross-linking. For this, a chitosan solution, that was previously neutralized with a glycerol phosphate disodium salt complex (the ionic cross-linker) was successfully prepared, to which genipin (the chemical crosslinker) was further added. The resulting hydrogels not only maintained the thermosensitive character that is typical of ionic cross-linking but also exhibited the improved mechanical properties and chemical stability that are usually associated with the presence of stronger covalent bonds. Moreover, the use of different loads of genipin allows some modulation of the network structure (and consequential beneficial behavior), which cannot be achieved by ionic cross-linking only. The study of samples that were prepared either without the glycerolphosphate salt complex or without genipin enabled the analysis of the effects that were generated by the presence of each cross-linker, demonstrating the benefits of the co-cross-linking method. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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