Chitosan Gelation Induced by the in Situ Formation of Gold

Biomacromolecules 2011, 12, 179–186

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Chitosan Gelation Induced by the in Situ Formation of Gold Nanoparticles and Its Processing into Macroporous Scaffolds Marıá J. Hortiguëla, Inmaculada Aranaz, Marıá C. Gutie´rrez, M. Luisa Ferrer, and Francisco del Monte* Instituto de Ciencia de Materiales de Madrid (ICMM), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Campus of Cantoblanco 28049 Madrid, Spain Received September 14, 2010; Revised Manuscript Received November 16, 2010

This work describes a simple synthetic route to induce chitosan (CHI) gelation by the in situ formation of gold nanoparticles (AuNPs). AuNPs were obtained by thermal treatment (e.g., 40 and 80 °C) of CHI aqueous solutions containing HAuCl4 and in the absence of further reducing agents. The CHI hydrogels resulting after AuNP formation were submitted to unidirectional freezing and subsequent freeze-drying via ISISA (ice-segregation-induced selfassembly) process for the preparation of CHI scaffolds. The study of AuNP-CHI scaffolds by SEM and confocal fluorescence microscopy revealed a morphological structure characteristic of the hydrogel nature of the samples subjected to the ISISA process. Interestingly, not only the morphology but also the dissolution and swelling degree of the resulting CHI scaffolds were strongly influenced by the strength of the hydrogels obtained by the in situ formation of AuNP. We have also studied the catalytic activity AuNP-CHI scaffolds in the reduction of p-nitrophenol. The negligible dissolution and low swelling degree obtained in certain AuNP-CHI scaffolds allowed them to be used for more than four cycles with full preservation of the reaction kinetics.

Introduction Looking to the next decades, nanoparticles and nanocomposites benefiting from the synergy between inorganic, organic, and biological entities will play a major role in the development of advanced functional materials for many different applications, being some of the most relevant among catalysis/biocatalysis and biomedicine.1 Assembling nanoparticles into macroscopic structures is challenging because the resulting materials would offer a desirable combination of high internal reactive surface area and straightforward molecular transport through broad “highways” leading to such a surface, which is of special relevance to any of the above applications.2 Therefore, we should be able not only to make nanostructures of any size and shape but also to assemble them in any form and to control their final structure at different space levels (e.g., hierarchically organized) so that their chemical nature and dimensions ensure accessibility to the inner interfaces.3 From a synthetic point of view, the progress in the preparation of advanced materials with nano- and microstructures and unprecedented performance depends largely on the core competence of materials chemists to design and develop novel synthetic strategies (mostly based on bottom-up techniques).4 Synthetic strategies that, in any stage of the synthesis, limit the use of chemical reagents (reducing agents, cross-linking agents, solvents, surfactants) that may eventually be difficult to eliminate from the reaction batch have lately attracted much attention for preparation of nanoparticles and nanocomposites useful in catalysis/biocatalysis and biomedicine.5 It is worth noting that the absence of undesired byproducts can be of help in either reducing nanoparticles poisoning in catalytic/biocatalytic reactions or preventing denaturation of biological entities (due to biocompatibility enhancement) in biomedical applications. * To whom correspondence should be addressed. E-mail: delmonte@ icmm.csic.es.

Besides the utilization of environmentally benign chemicals and solvents, the in situ preparation of nanocomposites into the required structure (e.g., spheres, nanospheres, capsules, or scaffolds, among others)6 is another key issue that merits important consideration. This is because the reduction or full elimination of processing steps that may eventually give rise to contamination can also be of help for the final performance of any catalytic or biomedical device. For instance, chitosan (CHI) metal nanocomposites have been obtained in the form of beads (e.g., micro- and nanoparticles)7 and films8 via the in situ formation of gold nanoparticles (AuNPs). In many of these cases, AuCl4- ions were reduced to zerovalent AuNPs by either the CHI itself9 or with the aid of the acetic acid used to dissolve it.10 Interestingly, in situ precipitation processes based on AuNP formation have never been used to obtain CHI hydrogels.11 Herein we report on a synthetic approach that induced CHI gelation upon the simultaneous formation of AuNPs. The process was based on the submission of a solution of CHI and HAuCl4 in acidified (0.16 M acetic acid) water (and in the absence of strong reducing agents9,12) to soft thermal treatments (e.g., 40 or 80 °C). The period of the thermal treatments ranged from 15 min to 5 h depending on the temperature (i.e., the higher the temperature, the shorter the treatment). It is worth noting that prolonged thermal treatments at 80 °C promoted the hydrogels to become viscous liquids again. Both AuNP-CHI hydrogels and solutions were subjected to the ISISA (icesegregation-induced self-assembly) process13 for the preparation of AuNP-CHI scaffolds, whose macroporous morphology strongly depended on the hydrogel strength of CHI provided by the in situ generated AuNPs. The reduction of Au salts after thermal treatment was followed by absorbance (UV-vis). AuNPs were studied by transmission electron microscopy (TEM) and X-ray photoelectron (XPS) spectroscopy. Scanning electron microscopy (SEM), confocal fluorescence microscopy, and swelling studies were used to assess the properties of the CHI scaffolds. The catalytic activity of AuNP-CHI scaffolds

10.1021/bm1010883  2011 American Chemical Society Published on Web 12/03/2010

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Biomacromolecules, Vol. 12, No. 1, 2011

for the reduction of p-nitrophenol in an excess of NaBH4 was monitored by UV-vis spectroscopy.

Experimental Part Preparation of CHI-Au Solutions. CHI (from Aldrich, batch no. 13604PC, av mol wt 817 KDa) was dissolved in acetic acid to obtain aqueous solution (CHI 2.5 wt %, acetic acid 0.2 M, pH 4.5) and mixed with another aqueous solution containing HAuCl4 · 3H2O. We achieved the preparation of CHI/HAuCl4 solutions having different HAuCl4 concentrations (0.2, 0.5, 1.0, and 2.0 mM for Au1, Au2, Au3, and Au4 samples, respectively) by mixing 1 mL of CHI solution to 0.250 mL of HAuCl4 · 3H2O 1.0, 2.5, 5.0, and 10 mM, respectively. The final CHI content and acetic acid concentration in the solution were 2.0 wt % and 0.16 M, respectively. The study of AuNP formation was carried out in samples thermal treated at 40 °C (T1) under dark conditions for times ranging from 15 min to 5 h. Hydrogels were obtained at ca. 6, 11, 40, and 150 min for Au4, Au3, Au2, and Au1 solutions, respectively. Hydrogels were dialyzed in 45 mL of distilled water to calculate the concentration of AcOH that remains entrapped within the hydrogel after AuNP formation by means of an acid-base titration of the dialysis solution with 5 mM sodium hydroxide aqueous solution. Au3 solutions were also thermal treated at 80 °C (T2) under dark conditions for times ranging from 20 to 120 min. ISISA Processing. The AuNP-CHI hydrogels subjected to the ISISA process were obtained from Au1, Au2, Au3, and Au4 solutions loaded in insulin syringes, thermal treated at 40 °C over 150 min, and thermal equilibrated at room temperature. The resulting AuNP-CHI hydrogels (AuNP1-CHIT1, AuNP2-CHIT1, AuNP3-CHIT1, and AuNP4-CHIT1) were unidirectionally immersed (at a nominal dipping rate of 2.7 mm/ min) in a cold bath maintained at a constant temperature of -196 °C.13 The frozen samples were freeze-dried using a ThermoSavant Micromodulyo freeze-drier. The resulting freeze-dried samples (e.g., AuNPCHI scaffolds) were monoliths with both the shape and the size of the insulin syringes. AuNP3-CHI hydrogels obtained from Au3 solutions thermal treated at 80 °C (T2) for times ranging from 20 to 120 min were also subjected to the ISISA process (AuNP3-CHIT220, AuNP3CHIT240, AuNP3-CHIT260, and AuNP3-CHIT2120), as described above. Acetic acid aqueous solutions of CHI (in the absence of Au salts) were also subjected to the ISISA process for the achievement of bare CHI scaffolds. Swelling Kinetics of AuNP-CHI Scaffolds. Swelling experiments were carried out by immersion of AuNP-CHI scaffolds in an excess amount of deionized water at 20 °C. The swollen samples were weighed at various time intervals. We calculated the swelling ratio by dividing the mass of absorbed water (obtained by subtracting the mass of dried scaffold from the mass of swollen hydrogel) by the mass of dried scaffold. Experiments were conducted in triplicate. Catalytic Activity of AuNP-CHI Scaffolds. As a model reaction, we chose the reduction of p-nitrophenol by sodium borohydride (NaBH4) to p-aminophenol. In a typical run, 0.3 mL of a NaBH4 solution (0.1 M) was added to 3 mL of a p-nitrophenol solution (0.05 mM). The pH was adjusted to 10 with NaOH. After stirring for 1 min, a given amount of AuNP-CHI scaffolds (ca. 3 mg) was added as catalyst. Therefore, the mass of AuNPs used in the reaction was ca. 9, 22.5, 45, and 90 µg in AuNP1-CHIT1, AuNP2-CHIT1, AuNP3-CHIT1, and AuNP4-CHIT1 scaffolds, respectively. We measured the process of the reduction by monitoring the extinction of solution at 400 nm as a function of time. Sample Characterization. Sample morphologies were investigated by SEM (Zeiss DSM-950 microscope). The morphologies of AuNPs were studied by TEM (200-KeV JEOL 2000 FXII microscope) and high-resolution transmission electron microscopy (HRTEM, EOL300FEG microscope), whereas the selected area electron diffraction (SAED) and energy-dispersive X-ray analysis (EDX) were used to study the crystallinity and composition of the sample, respectively. UV-vis spectrometry analyses were performed in a Variant Cary 4000 spectrophotometer. FTIR spectra were carried out in an FTIR Bruker IFS60v

Hortiguëla et al. spectrometer. Confocal fluorescence microscopy was performed with a Radiance 2100 (Bio-Rad) laser scanning system on a Zeiss Axiovert 200 microscope. Micrographs were taken in backscattering mode (the excitation wavelength was 415 nm). X-ray photoelectron spectroscopy surface analysis was performed in a VG ESCALAB 200R electron spectrometer equipped with a hemispherical electron analyzer and an Al KR (hν ) 1486.6 eV, 1 eV ) 1.6302 × 10-19 J) 120 W X-ray source. Cross-sectioned samples were carbon glued on 8 mm diameter stainless steel troughs mounted on a sample rod placed in the pretreatment chamber and degassed for 0.5 h prior to being transferred to the analysis chamber. The base pressure in the analysis chamber was maintained below 4 × 10-9 mbar during data acquisition. The pass energy of the analyzer was set at 50 eV. The binding energies were referenced to the binding energy of C1s core-level spectrum at 284.9 eV. Data processing was performed with the XPS peak program, and the spectra were decomposed with the least-squares fitting routine provided with the software with Gaussian/Lorentzian (90/10) product function and after subtracting a Shirley background. Atomic fractions were calculated using peak areas normalized on the basis of sensitivity factors provided by the manufacturer.

Results and Discussion UV-vis spectra depicted in Figure 1c show the evolution of the Au surface plasmon band at 525 nm in a CHI aqueous solution containing 1 mM HAuCl4 · 3H2O (Au3) subjected to 40 °C (T1) over different times. The increase in the surface plasmon intensity revealed the formation and growth of AuNPs. AuNP formation was studied for four Au concentrations (Au1, Au2, Au3, and Au4; see the Experimental Part). The band broadening and red shift observed in Au4 samples for thermal treatments over 240 min were indicative of large AuNPs and size heterodispersion (see inset in Figure 1d in comparison with insets in Figure 1a-c).14 Therefore, an optimum compromise (in terms of having a significant number of AuNPs with a narrow particle size distribution centered at ca. 5 nm, Figures 1 and 2a) was found for samples thermal treated over 150 min. The formation of AuNPs was further corroborated by HRTEM and SAED (Figure 2a,b). HRTEM showed the interplanar distance of AuNPs along the [111] zone axis (e.g., 0.235 nm). The SAED was typical of the crystal lattice of AuNPs with reflections belonging to (111), (200), (220), and (311) planes observed at d111 ) 0.238 nm, d200 ) 0.203 nm, d220 ) 0.143 nm, and d311 ) 0.124 nm.15 One of the most interesting processes taking place after HAuCl4 · 3H2O addition was that CHI solutions became gels at times depending on the Au salts concentration (Figure 3a). The red AuNP-CHI hydrogels obtained after thermal treatment at 40 °C over 150 min (Figure 3b) were submitted to the ISISA process13 to obtain AuNP-CHI scaffolds (AuNP1-CHIT1, AuNP2CHIT1, AuNP3-CHIT1, and AuNP4-CHIT1 in Figure 4a). The homogeneous distribution of Au0 throughout the entire section of AuNP-CHI scaffolds was confirmed by EDX (at SEM) and XPS (Figure 2c,d). XPS analysis of AuNP-CHIT1 scaffolds revealed the presence of Au 4f7/2 and Au 4f5/2 (at binding energies 83.8 and 87.4 eV, respectively), signals that are characteristic of Au0, whereas signals corresponding to Au+ ions were absent.16 The morphology of the macroporous structure was homogeneous throughout the entire monolith section, but macropores size and shape differed depending on the HAuCl4 · 3H2O concentration in the starting solution (see Figure 4b-f). Therefore, AuNP-CHI scaffolds prepared from 0.2 and 0.5 mM HAuCl4 · 3H2O dilutions (e.g., AuNP1-CHIT1 and AuNP2CHIT1) exhibited a cellular-patterned-like cross-section structure

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Figure 1. UV-vis spectra following the growth of the Au surface plasmon band resulting from the thermal treatment (at 40 °C) of acetic acid aqueous solutions having CHI 2.0 wt % and different HAuCl4 · 3H2O solutions having (a) 0.2, (b) 0.5, (c) 1, and (d) 2 mM concentrations. Thermal treatments were performed over 0 (black line), 15 (red line), 30 (blue line), 60 (green line), 90 (pink line), 120 (navy line), 150 (brown line), 240 (orange line), and 300 (dark yellow line, only represented for Au3) min. Insets show TEM micrographs of samples thermal treated at 40 °C over 240 min (bars are 60 nm).

(Figure 4b,c) and a micro-channeled-like longitudinal structure (Figure S1b of the Supporting Information), the latter resembling the unidirectional growth of ice crystals along the hydrogel. Meanwhile, AuNP-CHI scaffolds prepared from 1 and 2 mM HAuCl4 · 3H2O dilutions (e.g., AuNP3-CHIT1 and AuNP4CHIT1) exhibited a cellular-patterned-like structure in both cross(Figure 4e,f) and longitudinal sections (Figure S1c of the Supporting Information). It is well known that the macroporous structure of scaffolds obtained via ISISA is related to the viscosity of the solution (and eventually to the hydrogel strength) that is subjected to unidirectional freezing. Actually, CHI scaffolds prepared via ISISA from bare CHI solutions (e.g., in the absence of HAuCl4 · 3H2O and hence, nonjellified) exhibited a lamellar-patterned-like cross-section structure (Figure 4g) and a micro-channeled-like longitudinal structure (Figure S1a of the Supporting Information). Taking into account the fact that ISISA is an ice templating process, the influence that viscosity exerts on the macroporous morphology must be explained in terms of the capacity of ice crystals to form during the freezing process. Scaffolds preserved both the shape and size of the insulin syringes, where CHI solutions were placed before the thermal treatment and subsequent submission to the ISISA process, the preservation of which allows us to disregard any role played by freeze-drying on morphological changes. The capacity to form crystals upon freezing depends on multiple factors such as the temperature used for freezing, the freezing rate, and the solution concentration.17 In our case, freezing was carried out by immersion (at a constant dipping rate of 2.7 mm/min) of the samples in a liquid nitrogen bath, that is, at -196 °C. It is generally accepted that in samples submitted to the ISISA process, the zone of the probe that first comes into contact with liquid nitrogen exhibits no porosity at all after freeze-drying, and the material is dense. This feature is due to the formation of amorphous rather than crystalline ice (i.e., water can supercool when either a solution or hydrogel is submitted to -196 °C because of the presence of solutes)18 so that neither matter segregation nor formation

Figure 2. (a) HRTEM, (b) SAED, and (c) EDAX of AuNP3-CHIT1. (d) XPS of AuNP3-CHIT1 scaffolds showing the Au 4f7/2 and Au 4f5/2 doublet peaks characteristic of metallic gold at binding energies 83.8 and 87.4 eV, respectively.

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Figure 3. Picture of (a) AuNP-CHI gels having different HAuCl4 · 3H2O concentrations (from left to right; nil, 0.2, 0.5, 1, and 2 mM) obtained after thermal treatment at 40 °C over 150 min and (b) CHI sol and AuNP-CHI gels obtained after thermal treatment at 40 °C over (from left to right) 150, 150, 40, 11, and 6 min. Vials were turned upside down to emphasize their sol/gel nature.

Figure 4. (a) Picture of the monolithic AuNP-CHI scaffolds obtained from (from left to right) 0.2, 0.5, 1, and 2 mM HAuCl4 · 3H2O (AuNP1CHIT1, AuNP2-CHIT1, AuNP3-CHIT1, and AuNP4-CHIT1, respectively). SEM micrographs of (b) AuNP1-CHIT1, (c) AuNP2-CHIT1, (d,e) AuNP3-CHIT1, and (f) AuNP4-CHIT1 scaffolds. The SEM micrograph of a bare CHI scaffold without AuNPs is also included for comparison (g). Insets in (f) and (g) show details of the macroporous structures. Bars are (b,e-g) 200 µm, (c) 100 µm, (d) 1 mm, (f) 5 µm in inset, and (g) 20 µm in inset.

of any porous structure occurs. As soon as the probe comes into contact with the liquid nitrogen, an ice front appears running upward toward the nonimmersed portion of the probe. The

Hortiguëla et al.

Figure 5. (a) SEM micrograph of AuNP-CHI scaffolds prepared (via ISISA) from CHI aqueous solutions containing 1 mM HAuCl4 · 3H2O and thermal treated at 80 °C over 120 min (e.g., AuNP3-CHIT2120, bar is 200 µm). Inset shows a TEM micrograph of the AuNP obtained under these conditions (bar is 80 nm). (b) Picture of the AuNP3-CHI sol thermal treated at 80 °C over 120 min. (c) Picture of a CHI sol resulting from (1) thermal treatment at 80 °C over 120 min, (2) addition of 1 mM HAuCl4 · 3H2O, and (3) further thermal treatment at 80 °C over 20 min. Vials in parts b and c were turned upside down to emphasize their sol nature.

temperature at the ice front will obviously be higher than -196 °C and will depend on the ice front height above the liquid nitrogen level. In our case, the use of a constant dipping rate in the ISISA process allowed the maintenance of the ice front to a certain distance of the nitrogen level during most of the immersion process, which, ultimately, was crucial to obtain homogeneous porous structures throughout the entire monoliths (obviously, except in the tip that first comes into contact with liquid nitrogen which, in our case, was disregarded for further studies).17 We, and others, have also observed that the formation of small or large ice crystals upon freezing is not only related to the solute concentration but also, in the case that those solutes are oligomers or polymers, to its molecular weight; that is, the higher the concentration of solutes or the higher its molecular weight, the smaller the ice crystals. Recent studies on hydrogels have also described how the ice crystal size decreases with the increase in the hydrogel strength (or the cross-linking degree), the morphology of which reflects a transition from lamellartype to cellular-type morphology.19 In our case, this transition is actually reflected in Figure 4 for CHI scaffolds prepared from HAuCl4 · 3H2O salt concentrations ranging from nil to 2 mM. Interestingly, hydrogel strength affected not only the crosssectional morphology but also the longitudinal morphology, transitioning from microchanneled-type to cellular-type for HAuCl4 · 3H2O concentrations above 0.5 mM in the starting solution (Figure S1 in the Supporting Information.). The preparation of AuNPs in CHI solutions could also be carried out at 80 °C (T2), providing a similar growth pattern to that found at 40 °C except for the time scale, this ranging from 20 to 120 min rather than from 15 to 300 min. Actually, large and heterodisperse AuNPs (mean particle size was centered at ca. 13 nm and ranged from 5 to 20 nm; see TEM in inset of Figure 5a) were already obtained for thermal treatments at 80 °C over 120 min. For short thermal treatments (e.g., 20 min), CHI solutions became gels, and the morphology of the CHI scaffolds obtained after ISISA processing (e.g., AuNP3CHIT220) was equivalent to that shown for scaffolds obtained from hydrogels prepared at 40 °C (see Figure S2a in the Supporting Information). However, gel-to-sol transition (Figure 5b) occurred upon prolonged thermal treatments most likely as consequence of CHI rupture in shorter oligomers and the morphology of the resulting scaffolds resembled that of bare CHI scaffolds (compare Figures 4g and 5a). Actually, lack of gelation occurred when CHI was thermal treated at 80 °C for

CHI Gelation Induced by in Situ Formation of AuNPs

Figure 6. FTIR spectra of (a) AuNP3-CHIT220, (b) AuNP3-CHIT1, (c) AuNP1-CHIT1, and (d) bare CHI scaffolds.

120 min prior to the addition of HAuCl4 · 3H2O. A close inspection of the SEM micrographs obtained for bare CHI (Figure 4g) and for AuNP3-CHIT2 thermal treated over different times (Figures S2 in the Supporting Information and Figure 5a) further confirmed the relation between either the viscosity of the solution or the strength of the hydrogel subjected to the ISISA process and the morphology of the resulting scaffolds; that is, lamellar-type morphologies were obtained in scaffolds prepared from sols (e.g., bare CHI and AuNP3-CHIT2120 scaffolds shown in Figures 4g and 5a, respectively), intermediate morphologies were obtained in scaffolds prepared from soft gels (e.g., AuNP3-CHIT240 and AuNP3-CHIT260 scaffolds shown in Figures S2b and S2c), whereas cellular-type morphologies were obtained from strong gels (AuNP3-CHIT220 scaffolds shown in Figure S2a in the Supporting Information). After discussion of the scaffold morphology, we considered it of interest to study the mechanism governing both AuNP formation and CHI gelation. With regard to AuNPs formation and as mentioned in the Introduction, either CHI itself or in combination with the acetic acid used to dissolve it can reduce AuCl4- ions to zerovalent gold nanoparticles (AuNPs) by means of thermal or photochemical processes and in the absence of strong reducing agents (e.g., borohydride).9,12 Meanwhile, the well-known chelation of Au salts by amine groups induced CHI gelation.20 Actually, CHI did not become a gel in the absence of HAuCl4 · 3H2O (see Figure 3). The gelation time depended on the HAuCl4 · 3H2O concentration (Figure 3b). Those gels obtained after long treatments exhibited a red color characteristic of AuNPs. However, the pale-yellow color of those gels obtained over short treatments (e.g., 6 and 11 min) only anticipated AuNPs formation and confirmed the role of Au salts in CHI gelation. Interestingly, CHI remained a gel after AuNP formation (Figure 3a) so that one may speculate whether CHI gelation was related to the residual presence of Au salts after prolonged thermal treatments. This situation can be disregarded in our case because of the XPS data described above. Therefore, the preservation of the gel state of CHI after transformation of Au salts into AuNPs should somehow be ascribed to this reduction process but, in this stage of the work, this relationship was not yet specifically addressed. The study of CHI scaffolds obtained after the application of the ISISA process to solutions containing different HAuCl4 · 3H2O concentrations (from nil to 2 mM) by FTIR spectroscopy was useful in clarifying whether AuNP formation and CHI gelation are related (Figure 6). The FTIR spectrum of CHI has been discussed in previous papers,21 and the major


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peaks can be assigned as follows: 3411 cm-1 (O-H and N-H), 2929-2886 cm-1 (C-H), 1635 cm-1 (the coupling of CdO and N-H), 1560 cm-1 (N-H), 1410 cm-1 (the coupling of C-N and N-H), 1086 cm-1 (C-O), 897 cm-1 (ring). The FTIR spectra of every one of the scaffolds obtained upon Au reduction was similar in terms of both intensity and position of the bands (Figure 6a-c). However, the FTIR spectra of CHI scaffolds prepared in the absence of HAuCl4 · 3H2O exhibited three additional bands at 2963, 1711 (as a shoulder of the very intense band at 1610 cm-1), and 1262 cm-1 that resemble the presence of acetic acid (AcOH, Figure 6d).22 It is worth noting that acetic acid was used to dissolve CHI in water so that it will remain within the CHI matrix after the application of the ISISA process unless, in any stage of the process, it is somehow consumed. As described in previous works, AcOH can play the role of reducing agent in the formation of AuNPs.10 The AcOH concentration in the starting solution was 160 mM and decreased to 91, 61, 35, and 32 mM after AuNP formation in AuNP1CHIT1, AuNP2-CHIT1, AuNP3-CHIT1 and AuNP4-CHIT1 hydrogels.Therefore, AcOH consumption in the AuNPs reduction process resulted in the pH increase of the solution (i.e., the higher the HAuCl4 · 3H2O concentration, the higher the AcOH consumption) and, ultimately, induced CHI gelation. CHI gelation (rather than precipitation) requires a gradual pH rise not typically provided by regular base generators (e.g., ammonium carbonate, etc.).23 Otherwise (that is, if the pH rise is too sudden), the neutralization of the positive charges of CHI can make the system undergo coacervation-phase inversion as a consequence of uncontrolled CHI precipitation.24 The achievement of CHI hydrogels by controlled precipitation has lately attracted greater interest.11 For instance, CHI physical hydrogels have been obtained from aqueous acetic acid solutions of CHI also containing 1,2-propanediol. Under these circumstances, alcohol evaporation contributes to the elimination of both water and acetic acid, the release of which decreased the apparent charge density of CHI chains up to a critical value of gelation.11b Moreover, we have recently reported on the gradual increase in the pH resulting from the enzymatic decomposition of urea by urease that, in CHI aqueous solutions, also conduced to the formation of homogeneous CHI gels, the macroporous structure of which (after their submission to the ISISA process) was cellular-like as well.11c On the basis of these results, we can conclude that in our case CHI gelation occurred as consequence of two combined events: (1) CHI chelation by Au salts and (2) the pH rise resulting from the acetic acid consumption in the formation process of AuNPs. The application of the ISISA process to either sols or gels was crucial not only for the macroporous morphology of the resulting CHI scaffolds but also for their physicochemical properties. For instance, CHI scaffolds prepared from bare CHI solutions (i.e., in absence of HAuCl4 · 3H2O) were readily solubilized when placed in water. However, the solubility of AuNP-CHI scaffolds decreased with the increase in the HAuCl4 · 3H2O concentration in the starting solution (see Table 1). To corroborate this issue further, we also measured the equilibrium degree of swelling of AuNP-CHIT1 scaffolds prepared from different HAuCl4 · 3H2O concentrations. It is well known that swelling in hydrogels decreases for increased crosslinking densities.25 In our case, the equilibrium degree of swelling decreased with the hydrogel strength, the increase of which was directly related to the HAuCl4 · 3H2O concentration in the starting solution (Figure 7a); that is, the higher the HAuCl4 · 3H2O concentration, the lower the AcOH concentration remaining within the scaffold structure. Confocal fluorescence

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Hortiguëla et al.

Table 1. Dissolution (in wt %) of Bare CHI, AuNP-CHIT1, and AuNP-CHIT2 Scaffolds Soaked in an Excess Amount of Deionized Water at 20 °C over 10 (D10) and 60 (D60) min CHI AuNP1-CHIT1 AuNP2-CHIT1 AuNP3-CHIT1 AuNP4-CHIT1 AuNP1-CHIT220 AuNP2-CHIT220 AuNP3-CHIT220 AuNP4-CHIT220 AuNP3-CHIT240 AuNP3-CHIT260 AuNP3-CHIT2120

HAuCl4 (mM)

D10 (wt %)

D60 (wt %)

0 0.2 0.5 1.0 2.0 0.2 0.5 1.0 2.0 1.0 1.0 1.0

100 negligible negligible negligible negligible 10 10 6 5 9 17 53

100 15 (negligible)a 13 (negligible)a 12 (negligible)a 7 (negligible)a 22 20 13 11 18 33 100

a Samples were thermal treated at 40 °C over 10 days to promote the full evaporation of AcOH.

Figure 8. Confocal fluorescent microcopy micrographs of AuNP3CHIT1 and bare CHI scaffolds soaked in distilled water at 20 °C over 10 and 60 min.

Figure 7. (a) Time evolution of relative swelling degree (expressed as grams of solvent per gram of dry scaffold) obtained after immersion of AuNP1-CHIT1 (dark yellow circles), AuNP2-CHIT1 (red circles), AuNP3-CHIT1 (orange circles), and AuNP4-CHIT1 (blue circles) scaffolds in an excess amount of deionized water at 20 °C. (b) Evolution of relative swelling degree of the scaffolds (correspondence to symbols and experimental conditions remain as above) after full evaporation of AcOH by thermal treatment at 40 °C over 10 days. The swelling degree of a bare CHI scaffold thermal treated at 40 °C over 10 days is also represented for comparison (green circles).

microscopy confirmed that swelling in AuNP3-CHIT1 scaffolds was negligible, whereas bare CHI scaffolds (coming from sols rather than from hydrogels) were readily dissolved (Figure 8). AuNP3-CHIT2120 scaffolds exhibited a similar trend to that observed for bare CHI scaffolds. It is worth noting that because AcOH concentration was higher than HAuCl4 · 3H2O concentration in the starting solution, part of AcOH remained within the scaffold structure obtained after AuNP formation, CHI gelation, and freeze-drying (see above; 91, 61, 35, and 32 mM in AuNP1-CHIT1, AuNP2-CHIT1, AuNP3-CHIT1 and AuNP4-CHIT1 hydrogels, respectively). The capital role that remaining AcOH played in the solubility and swelling ratio of the resulting scaffolds was further corroborated by submission of the AuNP-CHIT1 scaffolds prepared from different HAuCl4 · 3H2O concentrations to a thermal treatment

(e.g., 40 °C, 10 days) that promoted the full evaporation of AcOH. As a consequence, the solubility and swelling ratio of thermal-treated scaffolds decreased, the final values of which were basically identical for every one no matter the HAuCl4 · 3H2O concentration used at the starting solution. (See Table 1 and Figure 7b.) Interestingly, elimination of AcOH from bare CHI scaffolds (thermal treated at 40 °C over 10 days) resulted in swelling ratios only slightly below than those found for AuNP-CHIT1 scaffolds. The remarkable structural stability gained by CHI scaffolds due to the presence of AuNPs allowed their use as catalytic substrates. As a model system that allows the evaluation of the catalytic performance, we studied the reduction of p-nitrophenol with NaBH4 in an aqueous medium and assisted by AuNPCHIT1 scaffolds as catalyst.26 The progress of the reaction was monitored spectrophotometrically as a result of the absorption in the UV-vis region of both the starting nitro compound (e.g., p-nitrophenol) as well as the product (e.g., p-aminophenol). In NaBH4 medium (pH ca. 10), the peak corresponding to p-nitrophenol (maximum at 317 nm) was red-shifted because of the formation of p-nitrophenolate ion (maximum at 400 nm). In the absence of any catalysts, the thermodynamically favorable reduction of the p-nitrophenol (E0 for p-nitrophenol/p-aminophenol ) -0.76 V and H3BO3/BH-4 ) -1.33 versus NHE) was not observed under the above-mentioned experimental condition; therefore, the peak due to the p-nitrophenolate ion (at 400 nm) remained unaltered (data not shown). The addition of AuNP-CHIT1 scaffolds in the reaction mixture catalyzed the reaction progress. UV-vis spectroscopy allowed us to monitor the reduction process (Figure 9a), that is, a decrease in the characteristic peak of p-nitrophenol at 400 nm while a new peak at 290 nm appeared due to the formation of p-aminophenol.26 The absorption spectra of p-nitrophenol were not disturbed by the presence of the AuNPs (

Chitosan Gelation Induced by the in Situ Formation of Gold

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