In Situ Precipitation of Amorphous Calcium ... - ACS Publications

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In Situ Precipitation of Amorphous Calcium Phosphate and Ciprofloxacin Crystals during the Formation of Chitosan Hydrogels and Its Application for Drug Delivery Purposes Stefania Nardecchia,† María C. Gutiérrez,*,† M. Concepción Serrano,† Mariella Dentini,‡ Andrea Barbetta,‡ M. Luisa Ferrer,† and Francisco del Monte*,† †

Instituto de Ciencia de Materiales de Madrid (ICMM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco. 28049 Madrid, Spain ‡ Dipartimento di Chimica, ″Sapienza″ Università di Roma, Piazzale Aldo Moro, 5. 00185 Roma, Italy ABSTRACT: The immobilization of more than one single substance within the structure of a biocompatible polymer provides multifunctional biomaterials with attractive and enhanced properties. In the context of bone tissue engineering, it could be of great interest to synthesize a biomaterial that simultaneously contains amorphous calcium phosphate (ACP), to favor calcium and phosphate precipitation and promote osteogenesis, and an antibiotic such as ciprofloxacin (CFX) that can, eventually, avoid infections resulting after surgical scaffold implantation. However, the co-immobilization of multiple substances is by no means a trivial issue because of the enhanced number of interactions that can take place. One of the main issues is controlling not only the diverse solid forms that individual substances can eventually adopt, but also the forces responsible for the self-organization of the individual components. The latter determines whether phase-separated structures or conjugated architectures are obtained and, consequently, may dramatically affect their functionality. Herein, we have observedby SEM, TEM, and solid-state NMRthat enzymatically-assisted coprecipitation of ACP and CFX resulted in phaseseparated structures. Thus, CFX crystals showed identical morphology to that obtained in the absence of ACP, but the size was smaller. Neither the size nor the morphology of ACP exhibited significant differences whether precipitated with or without CFX, but, in the former case, ACP was stabilized over a wider range of pH and temperature. Finally, by using this methodology and the ice segregation induced self-assembly process (ISISA), we have successfully co-immobilized ACP and CFX in chitosan-based scaffolds. Interestingly, the presence of ACP exerted significant control on the CFX release from these materials.

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INTRODUCTION Biomaterial science encompasses elements of medicine, biology, chemistry, and materials science.1 Tissue engineering and drug delivery are some of research fields where biomaterials have been most widely used.2 For both purposes, the production of suitable substrates involving the use of biocompatible and/or biodegradable materials and the processing of the components into a porous matrix of adequate morphology has been demonstrated to be crucial for the growth of cells and tissue as well as for the delivery and controlled release of any substance finally immobilized within the matrix structure. There has been quite a number of immobilized substances, ranging from active pharmaceutical ingredients (APIs) to inorganic nanoparticles and nanocomposites up to biological entities (from proteins to cells),3−6 with the general aim of providing enhanced functionality (hence, performance) to the resulting biomaterials. For instance, multiwalled carbon nanotube-based scaffolds containing both hydroxyapatite (HAp) crystals (as nuclei to promote further calcium and phosphate precipitation) and © 2012 American Chemical Society

rhBMP2 (a bone morphogenetic protein with osteoinductive properties) have exhibited a remarkable capability for the in vitro differentiation of myoblasts into collagen-expressing cells, because the combination of both entities can eventually provide a synergetic activity (hence, superior over that of the individual components).7 Solid-state chemistry is also gaining increased relevance in biomaterial science as a useful tool to control the diverse solid forms (e.g., polymorphs, pseudopolymorphs, salts, cocrystals, and amorphous solids) that immobilized substances can eventually adopt.8 It is well-known that the structure and composition of the crystalline nuclei, as well as the texture, the habit and aggregation, the size (of either the crystal or the aggregate), and the stability of intermediate phases, can significantly influence certain chemical and physical properties of a determined substance that may be relevant to the ultimate Received: July 4, 2012 Revised: October 5, 2012 Published: October 22, 2012 15937

dx.doi.org/10.1021/la3033435 | Langmuir 2012, 28, 15937−15946

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biocompatible character.29 Interestingly, the use of this enzymatically-assisted route (urease/urea system) allowed a gradual and smooth increase of the pH in the starting solution and the excellent control of the structural organization. In an attempt to elucidate the solid structures formed (from phaseseparated solids to cocrystals), we investigated by NMR and TEM the particular nature of CFX crystals and ACP coprecipitated in aqueous solutions containing urease and urea, but not CHI, as a simpler system where to avoid polymer interference in the characterization. Once the presence of cocrystals was disregarded, CHI hydrogels containing CFX crystals and ACP were ISISA processed for the achievement of 3D hybrid CHI-based scaffolds having both substances immobilized within the resulting macroporous structure. SEM was used to study the morphology of the macroporous scaffolds, whereas TEM and solid-state NMR allowed the characterization of embedded ACP nanoparticles. Finally, the capability of the resulting scaffolds as drug delivery systems was evaluated following the release of CFX by UV−vis spectroscopy.

performance of the biomaterial. For instance, the bioavailability and hygroscopicity of a determined API can vary greatly depending on its solid form.9 Moreover, the effectiveness of calcium phosphates to promote further calcium and phosphate precipitation (by dissolution and recrystallization) also depends on their original solid form.10,11 For instance, those nanocomposites containing amorphous calcium phosphate (ACP) would be of greater interest for biomineralizing purposes than those based on nanocrystalline HAp given the higher solubility of the former. This superior biomineralization capability of ACP makes it particularly attractive in dental applications.12 The best examples of solid-state chemistry to control the formation of a desired solid form can be found in nature.13 In biomineralization, structured organic surfaces are thought to play a key role in organic matrix-mediated deposition by lowering the activation energy of nucleation of specific crystal faces and polymorphs through interfacial recognition.13−16 Nonetheless, not only solid surfaces, but also soluble molecules and macromolecules, in addition to organic and inorganic ions, can have an important kinetic effect on crystallization by modifying the interactions with the nuclei so that different crystals, in terms of both polymorphism and habit modification, can ultimately be obtained.13−19 There are certain heterogeneous biomaterials (e.g., multifunctional biomaterials with more than one single immobilized substance like those on which we will focus our attention in this work) that belong to this category. In these cases, the increased number of interactive components tends to significantly enhance the diversity of emergent structures so that the design of synthetic processes starting from homogeneous solutions and capable of controlling all these interactions is critical. For instance, the forces responsible for the self-organization of individual components and those required for mutual co-assembly must be balanced. If the former are predominant, then phaseseparated structures will be synthesized, whereas domination of the latter can result in non-equilibrium conjugated architectures (e.g., cocrystals).20 This situation is exemplified by the hierarchical and hybrid nature of many biological minerals, which arise from an underlying synergy between the force fields of inorganic precipitation and biological organization.21−23 Biomimetic processes based on these principles serve as important archetypes in the synthesis of highly ordered materials across a range of length scales. We have recently demonstrated the suitability of an enzymatic process (based on the urease-assisted hydrolysis of urea) in combination with a unidirectional freeze−drying process (called ice segregation induced self-assembly,24,25 ISISA) for the preparation of chitosan (CHI) scaffolds which contain either inorganic26 or organic entities27 immobilized in a determined solid form, e.g., ACP and anhydrous ciprofloxacin crystals (CFX,28 a synthetic fluoroquinolone antimicrobial agent), respectively. In these publications,26,27 the enzymatically-assisted decomposition of urea by urease provided a smooth increase of the pH in the aqueous medium that was critical for the specific precipitation of either ACP or CFX concurrently with CHI gelation. Herein, we have applied this combined approach to the preparation of interesting multifunctional CHI scaffolds simultaneously containing ACP and anhydrous CFX crystals, where the former can remarkably promote osteogenesis by favoring calcium and phosphate precipitation processes, and the latter can avoid eventual infections resulting after surgical scaffold implantation. The polymer of choice was CHI because of its widely accepted

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Materials. Chitosan (CHI, Batch #06513AE, av. mol. wt. 617 KDa, deacetylation degree (DD) 88 ± 2), hydroxyapatite (HAp), urease (from Canavalia ensiformis (Jack bean) Type III, Sigma lot 115K7030, 45 units/mL), and urea were from Sigma Aldrich. Ciprofloxacin (CFX) was purchased from Normon Inc. (CIC2071) and doublelyophilized prior use. Buffered solutions were freshly prepared with K2HPO4 and NaOH from Sigma Aldrich. Water was distilled and deionized. Urease-Assisted Precipitation of ACP and of ACP and CFX in Water. A urea solution containing calcium-phosphate salts (e.g., 0.25 mL, urea 2 M, Ca5(PO4)3OH 0.03 M, HCl 0.3 M, pH 3.0) was added to an acetic acid solution (2.0 mL, 0.15 M) under vigorous stirring in an ice−cold bath. The pH was adjusted to 4.5 by addition of NaOH (10 M). The solution was maintained in a water bath at either 0 or 37 °C, prior to the addition of 0.175 mL of a freshly prepared urease aqueous solution (45 units/mL). The rise of the pH was monitored up to 8.0. The procedure followed for the simultaneous precipitation of both CFX and ACP was identical except that CFX was added as an acetic acid aqueous solution (0.25 mL, CFX 40 g/L, acetic acid 1 M) to the initial solution containing calcium and phosphate salts. Urease-Assisted Preparation of CFX-CHI Hydrogels. An aqueous solution of CHI (2 g, 2.83 wt % in acetic acid 0.15 M; pH 4.5) was mixed with an aqueous solution of CFX in acetic acid 1 M (0.25 mL, 40 g/L) and with an aqueous solution of urea (0.25 mL, urea 2 M, HCl 0.3 M, pH 3) under vigorous stirring in an ice-cold bath. Next, the resulting solution was mixed with 0.175 mL of a freshly prepared urease aqueous solution (45 units/mL for CFX-CHI1, 38.5 units/mL for CFX-CHI2, and 32 units/mL for CFX-CHI3) and stirred over 30 min. The resulting translucent liquid mixture was loaded into insulin syringes and aged for gelation (24 h at 37 °C up to reach a pH of ca. 8.0). Urease-Assisted Preparation of ACP-CFX-CHI Hydrogels. ACP-CFX-CHI1, ACP-CFX-CHI2, and ACP-CFX-CHI3 were prepared as described above for CHI-CFX hydrogels except that the urea solution also contained calcium-phosphate salts (e.g., 0.25 mL, urea 2 M, Ca5(PO4)3OH 0.03 M, HCl 0.3 M, pH 3.0). Rheological Measurements. Rheological experiments were carried out with a Bohlin CS10 stress-controlled rheometer using a concentric cylinder measuring system (C14). The measuring geometry was isolated by a Teflon septum to prevent solvent evaporation. About 3 mL of reaction mixture was charged. Preliminary stress−sweep experiments on completely formed gels were done in order to select a strain value in the range of linear viscoelasticity. Storage and loss moduli (G′ and G′′, respectively) were recorded as a function of time 15938


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during the course of gelation at 37 °C with a nominal strain of 2% and a frequency of 1 Hz. ISISA Processing of Hydrogels. The freshly gelled hydrogels were unidirectionally frozen by dipping (at 0.9 mm/min) the insulin syringes into a liquid nitrogen bath (77 K). The frozen samples were freeze−dried using a ThermoSavant Micromodulyo freeze−drier. The resulting scaffolds were monoliths with the shape and size of the insulin syringes used as containers. Plasma Treatment. CHI scaffolds were treated over 20 h in a commercial oxygen-plasma etching stripper (Tetra Pico, Diener Electronics) for elimination of the organic matter in mild conditions of temperature (e.g., 20 °C). The chamber was first evacuated to an ultimate pressure of about 0.12 mbar. Then, commercially available oxygen was leaked into the discharge chamber. The pressure was fixed at 0.4 mbar during the experiment. The plasma generator worked at a frequency of 40 kHz. The power used in the experiments was about 300 W. In Vitro Release Kinetic Experiments. A phosphate-buffered saline solution (PBS, 0.1 M, pH 7.4) was used as the medium for drugrelease tests. Similar amounts of every CHI/CFX scaffold were soaked in 5 mL of PBS solution at 37 °C to promote CFX release from the scaffold. The PBS solution was periodically removed (time intervals selected to fit sink conditions so that the drug concentration was always less than 10% of the saturation solubility in the release medium) and replaced by fresh PBS solution to continue the release test. The removed PBS solution was filtered through a 0.45 mm filter (Albeit-JCR regenerated cellulose) and analyzed by UV−vis spectrometry. The release of CFX was quantified by the measurement of the intensity of the absorbance peak at 276 nm (ε = 28 400 M cm−1). Background readings were obtained from CHI scaffolds without CFX. Released CFX was expressed as % of loaded CFX. Experiments were conducted in triplicate. Sample Characterization. Sample morphologies were investigated by scanning electron microscopy (SEM, Zeiss DSM-950 instrument) and transmission electron microscopy (TEM, 200-KeV JEOL 2000 FXII microscope). Energy-dispersive X-ray spectroscopy (EDX) for the elemental analysis and mapping was carried out using the accessory Apollo 10 integrated with FESEM Nova model NanoSEM 230 system working at 13 kV (the EDX mapping analysis collection time was 3500 s). UV−vis spectrometry analyses were performed in a Varian Cary 4000 spectrophotometer. Solid-state MAS NMR spectra were acquired using a BRUKER AV-400-WB spectrometer operating at 9.4 T and using a 4 mm triple channel probe. The working frequencies were 161.97 and 400.13 MHz for 31P and 1H NMR, respectively. The spinning frequency was set at 10 kHz in all cases. The sample weight was typically 50 mg. Solid-state 1H MAS NMR spectra were acquired with a π/3 pulse at 50 kHz, spectral widths of 35 and 50 kHz, and 5 s of relaxation time. Solid-state 31P MAS NMR spectra were acquired with π/6 single pulse at 50 kHz, spectral width of 35 kHz, and 10 s of relaxation time. The assignment of the chemical shifts in the 31P MAS NMR spectra was done using ammonium diphosphate (at −0.82 ppm) as secondary reference and referred to H3PO4 85% (at 0 ppm) as the primary one. The assignment of the chemical shifts in the 1H MAS NMR spectra was done using H2O (at 4.77 ppm) as secondary reference and referred to TMS (at 0 ppm) as the primary one.

transition from ACP to HAp is especially sensitive to the surrounding environment. In general, ACP hydrolyzes almost instantaneously into HAp, but, in the presence of other ions and macromolecules or under in vivo conditions, ACP may persist for appreciable periods and retain the amorphous state under some specific experimental conditions.30 ACP can also be either stabilized or destabilized in complex media because of the concurrent evolution of interactive components (e.g., pH, temperature, presence of polymers, etc.).31,32 In our case, the increase of pH promoted not only the precipitation of CFX, but also a significant increase of the viscosity that ended with CHI gelation. The formation of this gel introduces an additional difficulty for isolation (and hence, characterization) of any eventual precipitate. This is why herein we first induced the precipitation of calcium-phosphate salts in simple aqueous solutions (in both the absence and the presence of CFX) by decomposition of urea at 4 and 37 °C in the presence of urease. As a general trend, ACP and HAp precipitates exhibit a quite different morphology. ACP is granular-like (given its amorphous character) and consists of “Posner clusters” of approximately 1.0 nm in diameter, which aggregate randomly forming large spherical particles of 20−300 nm with tightly bound water residing within the interstices,11,32,33 whereas HAp crystals are micrometric platelets typically arranged in a flowerlike fashion. In absence of CFX, TEM micrographs of the precipitates revealed that ACP could only be stabilized when the pH was maintained below 6.5 at 4 °C, but not at 37 °C (Figure 1). Otherwise, flower-like HAp crystals were obtained.

RESULTS AND DISCUSSION Enzymatically-Induced Precipitation of ACP and CFX in Aqueous Solutions. As mentioned in the Introduction, the driving force inducing not only precipitation of CFX crystals and calcium-phosphate salts, but also CHI gelation, was the increase of pH resulting from urea decomposition in the presence of urease. In the particular case of calcium-phosphate salts, our main concern was to determine the experimental conditions necessary to stabilize ACP because of the strong tendency of this non-crystalline form to become HAp by dissolution and recrystallization. It is worth noting that

Figure 1. TEM micrographs of granular- and platelet-type precipitates of calcium phosphate obtained via enzymatically-induced decomposition of urea in aqueous solutions at 4 and 37 °C. The final pH ranged from 6.5 for (a,c) to 7 for (b) and up to 7.5 for (d).

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The selected area electron diffraction (SAED) of ACP did not reveal any crystallographic ordering, while that of HAp exhibited the typical reflections for crystalline hydroxyapatites synthesized at low temperatures.34 The very specific experimental conditions required for the achievement of ACP revealed how crucial the use of urease was for urea 15939


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decomposition. It is worth noting that, in the absence of urease, urea decomposition occurs at temperatures close to 90 °C. Under these conditions of high temperature and sharp pH increase, ACP is difficult to isolate because HAp tends to readily appear in the first precipitates.34 Within this context, the enzymatically-induced precipitation of CFX in CHI solutions has been described in one of our recent works,27 but, at that time, we did not yet study the precipitation of CFX in CHI solutions that also contained calcium-phosphate salts. For simplicity and as mentioned above for ACP, we first studied the precipitation of CFX and calciumphosphate salts in aqueous solutions (without CHI). The solidstate 31P MAS NMR spectrum of this precipitate exhibited a broad band at 2.8 ppm that is typically ascribed to PO43− groups in ACP precipitates and low-crystallinity HAp (Figure 2a).35−39 The 1H MAS NMR spectrum of the precipitate revealed an intense band at 5.1 ppm that is typically ascribed to weakly bound water (Figure 2b). Weakly bound water could be part of not only hydrated ACP, but also hydrated CFX, the hygroscopic character of which is well-known.27 Moreover, lowcrystallinity HAp can exhibit this band as well, although it is typically accompanied by a narrow and intense peak centered at 0 ppm (Figure 2c) that correspond to the hydroxyl groups that form part of the crystalline structure of HAp structure (Ca5(PO4)3(OH)).38,40,41 The presence of a low-intensity peak at ca. 0.2 ppm has been observed in octacalcium phosphate (Ca8H2(PO4)·6.5H2O), a phase that lacks structural hydroxyl groups, but may contain disordered ones.35 The 1H MAS NMR spectrum of the precipitate shown in Figure 2b revealed the presence of a low-intensity peak at ca. 0.5 ppm that could be ascribed to disordered hydroxyl groups, as they were deshielded as compared to those of HAp (e.g., at 0 ppm). On the basis of these results, we concluded that the presence of HAp was unlikely. Nonetheless, we decided to further investigate the nature of the calcium-phosphate precipitates by electron microscopy techniques (e.g., TEM and SEM). Neither the precipitation of CFX nor its morphology (e.g., needle-like) was apparently modified by the presence of calcium-phosphate salts (Figure 3). TEM micrographs also showed the presence of granular-like aggregates that resembled those observed in aqueous solutions (Figure 3). Interestingly, the presence of CFX crystals helped to stabilize ACP over a wider range of not only pH (up to 7.5 at 4 °C, left column in Figure 3), but also temperature (up to 37 °C for pHs reaching 6.6, right column in Figure 3) than those used in the absence of CFX crystals (Figure 1). Obviously, precipitations carried out at increased temperatures and/or higher pHs resulted in larger ACP particles (compare ACP sizes in Figures 3 and 4). Precipitation of calcium-phosphate salt preferentially occurred on the CFX needle-like crystals because carboxylic groups acted as Ca2+-binding sites.42 ACP stabilization was hypothesized to be a consequence of its preferential growth on the CFX needle-like crystals, the surface of which (as typically occurs with any other substrate in mineralization processes)8,13,14 restricted ACP growth and stabilized this intermediate calcium-phosphate precipitate. SEM combined with energy-dispersive X-ray spectroscopy (EDX) allowed analysis of the spatial distribution of elements that are forming these structures. Thus, carbon, oxygen, and fluoride were the only elements detected at the CFX needle crystals in conformity with CFX composition, while calcium and phosphorus were detected only at the granular precipitates deposited on the CFX crystals (Figure 4). The Ca/P ratio was

Figure 2. Solid 31P MAS NMR (a) and 1H MAS NMR (b) spectra of ACP precipitated on CFX crystals in the presence of urease and urea. Inset shows the absence of peaks in the 1H MAS NMR spectrum at 0 ppm. The solid 1H MAS NMR spectrum of HAp crystals is also included for comparison (c).

Figure 3. TEM micrographs of the granular-type calcium-phosphate precipitates and the needle-like CFX crystals that were obtained via enzymatic decomposition of urea in aqueous solutions at 4 °C and pH 7.5 (left column) and at 37 °C and pH 6.6 (right column).

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Figure 4. SEM micrograph (left panel) and EDX elemental analysis (right panel) to confirm the presence of granular-type calcium-phosphate precipitates on a needle-like CFX crystal. Precipitates were collected at 37 °C and at pH 8.0.

inspection of the plot of G′ and G′′ over time revealed that, after the crossover, both G′ and G′′ experienced a second sigmoidal transition in which transparent gels became turbid. The occurrence of this second sigmoidal transition was ascribed to the pH-induced segregation of the originally homogeneous gel into polymer-rich and polymer-poor domains with sizes within the range of visible wavelengths (Figure 5d), the formation of which also explains the increase of both the elastic (G′, ascribed to the polymer-rich phase) and the viscous (G′′, ascribed to the polymer-poor phase) moduli of the sample. The resulting hybrid hydrogels, by means of the enzymatically-assisted gelation and precipitation, were unidirectionally frozen (at 77 K) and subsequently freeze−dried for the achievement of cryogel nanocomposites whose macroporous structure resulted from the ISISA process described elsewhere.24,25,45−50 Briefly, ice formation (hexagonal form) causes every solute originally dissolved/dispersed in the hydrogel to be segregated from the ice phase. After freeze−drying, the empty areas where ice crystals were originally located corresponded to macropores, while the scaffold structure is supported by the matter accumulated between adjacent ice crystals. This process has demonstrated its suitability for the preparation of macroporous monoliths and fibers of different compositions (inorganic, organic, hybrid, and even composites) by freeze− drying the respective hydrogels and aqueous colloidal suspensions.26,27,51−54 Moreover, the process is highly biocompatible, as it begins from an aqueous suspension and runs in the absence of further chemical reactions, thus avoiding potential complications associated with byproducts or purification procedures. This biocompatible character has allowed immobilizing proteins, liposomes (e.g., membrane structures that mimics that of cells), and even bacteria within the resulting scaffolds.55−57 After ISISA processing, we investigated whether the incorporation of CFX and calciumphosphate salts modified the morphology of the scaffolds resulting after the ISISA process. For this purpose, we compared the morphology of a bare CHI scaffold (containing neither CFX nor calcium-phosphate salts), a CHI scaffold containing CFX (e.g., CFX-CHI1), and a CHI scaffold containing both CFX and calcium-phosphate salts (e.g., ACPCFX-CHI1). In every case, the scaffolds exhibited a similar cellular patterned-like cross-section structure (Figure 6a,c,e).

ca. 1.5, in agreement with the idealized molecular formula of ACP either anhydrous or hydrated (e.g., Ca9(PO4)6 or Ca9(PO4)6·nH2O, respectively) that is typically obtained at pHs around 6.6.43 The absence of foreign elements in both needle-like crystals and granular precipitates confirmed that, despite the heterogeneity of the aqueous medium, precipitation resulted in well-defined phase-separated structures rather than in any sort of non-equilibrium conjugated architectures or cocrystals. Enzymatically-Induced Precipitation of ACP and CFX Concurrent to CHI Gelation. At this stage, we focused on the study of the enzymatically-induced precipitation of CFX and calcium-phosphate salts in an aqueous CHI solution, according to the Experimental Section. In this case, the in situ slow generation of base provided by the enzymatically-assisted hydrolysis of urea promoted a gradual increase in pH that caused not only the precipitation of CFX and calciumphosphate salts, but also the gelation of chitosan. On the basis of the above-observed mutual influence that CFX and calcium-phosphate salts have on their respective precipitation, we first studied whether the gelation kinetics of bare CHI solutions was modified by the presence of either CFX or calcium-phosphate salts or both. For this purpose, we monitored the elastic (G′) and the viscous (G′′) moduli over time. As a general trend in gelation processes, G′′ is originally larger than G′ because the viscous properties dominate in the liquid state. Upon gelation, G′′ increases, but G′ increases more because the formation of the cross-linked (either physical or chemically) network that characterizes the gel state makes the elastic properties dominate over the viscous ones. Under these circumstances, G′ and G′′ data will eventually cross over time and the time at which this crossover occurs is defined as the gel point.44 The gelation times thus calculated showed that, as compared to bare CHI, the presence of CFX delayed gelation. Interestingly, the simultaneous presence of CFX and calciumphosphate salts caused a further delay in gelation (Figure 5). Despite the common use of G′ and G′′ crossovers for the determination of gel times, it is generally assumed that multifrequency experiments provide more accurate data. In our case, the gel time obtained from G′ and G′′ crossover was in agreement with that obtained from the plot of loss tangent versus time at different frequencies (inset in Figure 5a). A close 15941


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Figure 6. SEM micrographs of CHI1 (a,b), CFX-CHI1 (c,d), and ACP-CFX-CHI1 (e,f) scaffolds. Arrows in (d) indicate some representative CFX crystals entrapped within the CHI matrix.

folds.41 It is worth noting that a low-intensity peak at ca. 0 ppm also appeared. As mentioned above, this chemical shift is typically ascribed to well-ordered hydroxyl groups forming part of the crystalline structure of HAp.38,40 Considering its low intensity, one could hypothesize the presence of a minor fraction of HAp eventually evolving from ACP. However, this issue could not be confirmed at this stage considering that this peak also appeared in CHI scaffolds without calcium-phosphate salts (both with and without CFX; see Figure 7c,d). The above NMR results were thus inconclusive for the unambiguous assignment of the nature of the calciumphosphate precipitate so that, as above, we focused on electron microscopy techniques for the clarification of this issue. Thus, a close inspection to those scaffolds that contained CFX allowed the observation of crystals (Figure 6d,f). The morphology of these crystals was needle-like resembling that of CFX crystals. Interestingly, their size was significantly smaller than that of those found in solution and, even, decreased further in ACPCFX-CHI1 scaffolds as compared to CFX-CHI1 ones. This diminution of the size could be ascribed to the tendency of calcium-phosphate salts to precipitate on the CFX crystal surface (Figure 3). However, the direct observation of ACP was not possible in the CHI scaffold containing calcium-phosphate salts because the size of this precipitate was too small to provide any visual change in the morphology of CHI after its fully

Figure 5. Elastic (G′, blue triangles) and viscous (G′′, red circles) moduli of aqueous solutions containing bare CHI (a), CHI and CFX (b), and CHI, CFX, and calcium-phosphate salts (c) over time. Inset in (a) shows the loss tangent versus time for different constant shear frequencies in an aqueous solution containing bare CHI. (d) Scheme representing the transition from sol to gel upon the rise of pH over time and the visual changes observed at the different stages.

As in the previous section, we used solid-state 31P MAS NMR and 1H MAS NMR spectroscopy to investigate whether ACP was the preferred calcium-phosphate precipitate formed in CHI scaffolds that also contained CFX. As mentioned above, the peak at 2.6 ppm in the solid-state 31P MAS NMR spectrum was attributed to PO43− groups (Figure 7a).35 Broad peaks are typically indicative of the low-crystallinity character of the calcium-phosphate precipitate, but this signature does not allow by itself to discern unambiguously between HAp (for instance, nanocrystals with low crystallinity) and ACP. Meanwhile, the 1 H MAS NMR spectrum depicted in Figure 7b exhibited a broad peak centered at ca. 5 ppm that revealed the presence of adsorbed and/or structural water in ACP-CFX-CHI1 scaf15942


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Figure 7. Solid 31P MAS NMR (a) and 1H MAS NMR (b) spectra of ACP-CFX-CHI1 scaffolds. The solid 1H MAS NMR spectra of CFX-CHI1 and CHI1 scaffolds are also included for comparison (c,d, respectively).

entrapment within the polymer matrix.26 We hypothesized that CHI elimination would allow the isolation of the precipitate and its subsequent observation by TEM. Unfortunately, approaches based on thermal treatment were not suitable because any calcium-phosphate precipitate would promptly transform into crystalline apatites.26 Thus, the scaffolds were exposed to an oxygen plasma treatment. Low-temperature plasma treatments have been progressively applied to activate the outermost polymer surfaces, likewise, not to affect its structural dimensions.58 Surface modification occurs upon exposure to reactive plasma over a few seconds by the formation of ionized species and free radicals that interact with an organic surface by forming oxidative groups. However, it has been found that longer treatment times can result in further deterioration of the organic matter.59 In our case, treatments prolonging over more than 10 h at room temperature resulted in the elimination of all the organic matter (both CHI and CFX). After an exhaustive observation of large fields of view by TEM, we only found granular structures (Figure 8) that were similar to those observed in Figures 1a and 3 and, hence, ascribed to ACP. We could thus conclude that ACP is the main calcium-phosphate precipitate in these samples. Nonetheless, it is fair to recognize that the presence of a minor fraction of HAp evolving from ACP (e.g., as small crystallites of about 3−5 nm of ACP spheres, with several crystallographic units of HAp in depth)32 cannot be absolutely ruled out by this technique. Actually, we have recently reported how difficult is to obtain pure crystalline phases by electrodeposition of calciumphosphate salts even though all these crystalline phases were more stable than ACP.25 Nonetheless, it is worth noting that

Figure 8. TEM micrograph of ACP immobilized in ACP-CFX-CHI1 scaffolds after plasma treatment and elimination of the organic matter.

the presence of more than one single phase was not as evident in this case as it was in our previous work. Kinetic Release of CFX from CHI Scaffolds with and without ACP. We finally studied the kinetic release of CFX from CFX-CHI1 and ACP-CFX-CHI1 scaffolds. The plot representing the CFX release from both scaffolds exhibited two well-differentiated kinetics: burst-type for a certain percentage of the immobilized CFX, followed by a sustained release for the remaining one (Figure 9). In previous work,27 we ascribed this behavior to the anhydrous character conferred to the CFX crystals by the freeze−drying process used for scaffold preparation (e.g., ISISA). These anhydrous CFX crystals can be both dissolved and hydrated in the presence of water. Interestingly, the solubility of anhydrous and hydrated crystals is quite different, high for the former and low for the latter.27,60 Thus, the dissolution of anhydrous CFX crystals could be 15943


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the anhydrous CFX crystals decreased from ACP-CFX-CHI3 to ACP-CFX-CHI2 up to ACP-CFX-CHI1 (Figure 9).

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CONCLUSIONS We have demonstrated that the enzymatically-assisted coprecipitation of ACP and CFX in aqueous solutions resulted in phase-separated structures rather than in conjugated architectures. The morphology of CFX crystals was not apparently modified by the presence of ACP, although crystal size was eventually smaller in the presence of the latter. Interestingly, the stability of ACP was extended over a wider range of pH and temperatures in the presence of CFX crystals as compared to bare aqueous solutions. We have also promoted the coprecipitation of ACP and CFX by urease-assisted hydrolysis of urea in aqueous solutions containing CHI that allowed the preparation of hybrid scaffolds containing both anhydrous CFX crystals and ACP by the application of the ISISA process. Finally, we have demonstrated that ACP-CFXCHI scaffolds are suitable substrates for drug delivery and controlled release purposes. These materials may offer interesting perspectives as multifunctional biomaterials for bone tissue regeneration, as they combine multiple substances in the scaffold structure that may promote osteogenesis while preventing bacterial infections after surgical scaffold implantation.

Figure 9. Representative kinetic release of CFX from CFX-CHI1 (solid circles), ACP-CFX-CHI1 (open circles), ACP-CFX-CHI2 (open squares), and ACP-CFX-CHI3 (open diamonds) scaffolds.

described as a three-stage process consisting of (1) fast dissolution of the outer layers of the anhydrous crystals, (2) hydration of the remaining anhydrous crystals and, thus, formation of hydrated ones, and (3) slow dissolution of the hydrated crystals.27 This dissolution mechanism corresponded well with the kinetics observed in Figure 9 (e.g., burst release first, followed by a more sustained one onward), and it also explained the increase of the CFX percentage released in a burst-type fashion in ACP-CFX-CHI1 as compared to CFXCHI1 considering that the ratio of crystal external surface versus internal core decreases along with the size of the anhydrous CFX crystal. To corroborate this issue, we prepared ACP-CFX-CHI scaffolds decreasing the urease concentrations used for precipitation and gelation (e.g., ACP-CFX-CHI2 and ACP-CFX-CHI3). The use of different urease concentrations provided similar CHI macroporous structures irrespective of the presence of CFX (Figure 10). However, the decrease of the urease concentration delayed CHI gelation (by slowing down the pH rise) and, hence, allowed CFX crystals to grow further (Figure 10). Interestingly, there was a certain decrease of the percentage of CFX released in a burst-type fashion as the size of

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from MINECO (Project Numbers MAT2009-10214, MAT2011-25329, MAT201234811 and PIE201060I017) and from the European Union Seventh Framework Programe (FP7/2007-2013) under grant agreement no 263289 (Green Nano Mesh). S.N. acknowledges CSIC for a JAE-Pre fellowship. M.C.S. acknowledges MINECO for a Juan de la Cierva research contract. Fernando Pinto (from the Instituto de Ciencias Agrarias-CSIC) and Mariá José de la Mata (from SIdI-UAM) are acknowledged for their kind assistance with SEM and soli-state NMR studies, respectively.

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Figure 10. SEM micrographs of ACP-CFX-CHI2 (a,b) and ACPCFX-CHI3 (c,d) scaffolds. 15944


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