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AIEgen quantitatively monitoring the release of Ca2+ during swelling and degradation process in alginate hydrogels
Materials Science & Engineering C 104 (2019) 109951
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
AIEgen quantitatively monitoring the release of Ca2+ during swelling and degradation process in alginate hydrogels
T
Javad Tavakolia, Esther Laisaka, Meng Gaob, Youhong Tanga,
⁎
a
Institute for Nanoscale Science and Technology, Medical Device and Research Institute, College of Science and Engineering, Flinders University, South Australia 5042, Australia b National Engineering Research Center for Tissue Restoration and Reconstruction, School of Material Science and Engineering, South China University of Technology, Guangzhou 510006, China
ARTICLE INFO
ABSTRACT
Keywords: Alginate hydrogels Aggregation-induced emission (AIE) Calcium release Swelling Degradation
Alginate-based hydrogels are extensively used for different biomedical applications. While the swelling and degradation of alginate-based hydrogels affect their structure-property relationship, many studies employed gravimetric analysis to characterize the swelling-degradation process. Accurate or not, this traditional method is difficult to be consistently performed with minimized errors, especially at the late stage of the process. For the first time, this study introduced a reliable, accurate and cost-effective method to minimize the human-sourced errors during repetitive measurement of swelling and degradation of alginate-based hydrogels based on Ca2+ specified aggregation-induced emission fluorogen technology. This study provides an approach for characterization of different properties of alginate-based tissue engineered scaffolds. The established relation between the changes in released Ca2+ into the swelling environment and its relative intensity identified the potential application of the proposed method for prediction of swelling and degradation behaviour in alginate-based hydrogels.
1. Introduction Recent evolution in the design of new biomaterials has been focused on naturally-driven materials that are able to mimic the function of extracellular matrix of tissues and regulate the host responses [1–6]. Owing to the inherent biocompatibility, alginate is a natural material with wide range of applications in medical fields [7,8]. Alginate is a water-soluble anionic polymer that extracted from brown algae, i.e., seaweed by treatment with alkali solution [9]. From a structural point of view, alginate is a polysaccharide copolymer of (1-4)-β-D-mannuronic acid and (1-4)-α- L-guluronic acid, where their ratio depends on its natural source [10]. When crosslinked with divalent cations, for example Ca2+, a biocompatible alginate hydrogel with structural similarity to the extracellular matrix of living tissues is formed that allows wide applications in tissue engineering [11], delivery of bioactive agents and macromolecular pharmaceutics [12], cell transplantation [13] and wound healing [14,15]. The swelling and degradation (SD) of alginate-based hydrogels that affect their biomedical applications can be controlled by partial oxidation using sodium periodate, change in crosslinking duration (time) or concentration and regulating the molecular weight distribution
⁎
[16,17]. Since the biocompatibility of alginate hydrogel is likely to decrease at high degrees of oxidation [17], the use of low molecular weight sodium alginate and optimized concentration of crosslinking agent is a proper approach that offers control over the SD rates. No matter which of the strategies are used, the most frequent practical approaches to characterize the SD rates are gravimetric methods, measuring the weight change in hydrogel, as shown in Eq. (1) or analysing the viscosity of swelling environment over time [18].
SD Changes =
Mt
M0 M0
× 100
(1)
where, M0 and Mt are weights at the initial and time t, respectively. Moreover, several studies employed the DSC analysis and mechanical properties measurement to indirectly address the SD process, since a change in glass transition temperature and ultimate strength of hydrogel is expected [18,19]. Accurate or not, the traditional methods are difficult to be consistently performed with minimized errors, especially at the late stage of SD process. Since the fluorescence (FL) properties of aggregation-induced emission fluorogens (AIEgens) is improved by the restriction of intramolecular rotation (RIR) in aggregated state, they have been widely
Corresponding author. E-mail address: [email protected] (Y. Tang).
https://doi.org/10.1016/j.msec.2019.109951 Received 26 November 2018; Received in revised form 4 June 2019; Accepted 5 July 2019 Available online 07 July 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 104 (2019) 109951
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Fig. 2. Change in the weight and release of Ca2+ ions during SD process of sodium alginate hydrogels crosslinked for (a) 1 and (b) 5 min. The vertical red dot lines identify the swelling (region II), equilibrium (region III) and degradation (region IV) phases. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. (a) Dynamics of relative FL intensity change with SA-4CO2Na (0.5 mM) in CaCl2 solution (100 mM), inset: Chemical structure of SA-4CO2Na and (b) the relative FL intensity change as the function of Ca2+ concentration with 0.5 mM of SA-4CO2Na.
the FL intensity of the collected swelling environment was measured by addition of the AIEgen, simultaneously.
used for fabrication of advanced biomaterials, cell imaging, drug delivery and photodynamic therapies [20–26]. The characters of high emission efficiency in the aggregated state, excellent photo-stability, and high sensitivity make AIEgens suitable for biomedical applications. Currently, the application of AIEgens in tissue engineering and regenerative medicines has become a hot topic since provides a simple framework for better understanding of the interaction between host tissue and biomaterials, resulting in design of more efficient implants [27]. Based on the recent progress reporting wide range of applications for AIEgens [28–32]; here, a new approach for real time monitoring of release of Ca2+ during the SD process of sodium alginate-based hydrogels is presented. A recent study identified that the AIEgen, SA4CO2Na can be well dispersed in an aqueous solution with a very weak fluorescence emission. However, the AIEgen could form highly emissive aggregates in the presence of Ca2+ through electrostatic and chelating interactions [33]. In the current study, this AIEgen, SA-4CO2Na, was used for real-time detection and quantification of Ca2+ ions that are released during the progression of SD process. To demonstrate this concept, low viscosity sodium alginate and 100 mM CaCl2 solutions were used for preparation of droplet alginate hydrogels, as described before [34]. The SD properties of sodium alginate hydrogels were determined by measuring the changes in hydrogel's weight. Meanwhile,
2. Materials and method 2.1. Materials and reagents Sodium alginate (Alginic acid sodium salt from brown algae, Low viscosity, A1112) and CaCl2 (anhydrous, granular, C1016) were purchased from Sigma-Aldrich. The AIEgen, SA-4CO2Na, was a gift from our collaborator laboratory at Hong Kong University of Science and Technology, Hong Kong, China. 2.2. Sample preparation A stock solution of SA-4CO2Na in distilled water with a concentration of 50 mM was prepared and then diluted to 0.5 mM solution and kept in a sealed glass bottle. The solution was wrapped in aluminium foil and stored in a refrigerator under 4 °C for further use. Sodium alginate (4% w/w), NaCl (3% w/w) and CaCl2 (100 mM) aqueous solutions were prepared by mixing appropriate amount of powder in distilled water at room temperature and stirring (using magnet stirrer) for 5 h and 5 min, respectively. A 3 mL syringe (gauge 18) was used for preparation of sodium alginate hydrogel beads by injection of sodium alginate solution (4% w/w) into the CaCl2 solution (100 mM) under 2
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Fig. 3. Schematic drawings of SD process in sodium alginate hydrogels (I) early stage of swelling, (II) swelling region where the diffusion of water into the hydrogel network results in breakage of temporary junctions (III) repulsion of fixed negative charges (at final stage of swelling and equilibrium states) increases the uptake of water and resulted in enlargement of lattice size and (IV) degradation of hydrogel network proposed by dissociation of chains at the chelate structure. Alginate chains including chelate structures, calcium ions and water molecules denoted by orange-black, green and blue circles, respectively and (a) the corresponding Ca2+ release and weight change during Regions I-IV. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
slow stirring (50 rpm). Beads were extracted from CaCl2 solution after 1 and 5 min of crosslinking and rinsed with distilled water for 1 min.
Briefly, a standard cuvette (3 mL volume) was filled with 2 mL of SA4CO2Na aqueous solution (0.5 mM). A sodium alginate bead was placed in distilled water or NaCl solution. Then samples including 0.5 mL of swelling environment were collected at each time point using a sampler (1000 μL) and injected into a cuvette that was filled with SA-4CO2Na aqueous solution. All cuvettes were allowed to stand for 30 min before FL data collection (Spectra and intensity). To evaluate the effect of sonication on the release of Ca2+ ions from sodium alginate hydrogels, wet beads (crosslinked for 5 min) with approximately similar initial weights (3.6 ± 0.02 mg) were placed in a cuvette including 2 mL of SA-4CO2Na aqueous solution (0.5 mM). A continuous sonication (sweep mode) was applied to the sample for 10 min time intervals using a sonication bath (MRC) at room temperature and the FL intensities were measured, subsequently. All sample preparation and relevant measurements were performed using sealed containers (cuvettes) to avoid evaporation.
2.3. Characterization The SD process was measured using gravimetric method by measuring the weight of beads that were placed in distilled water and NaCl solution at different time points. Both dry (dried for 24 h at room temperature after crosslinking with CaCl2 Solution) and wet beads (immediately used after crosslinking in CaCl2 solution) were used. The SD process for wet samples was performed immediately after preparation of samples (sodium alginate beads). The detection of Ca2+ was conducted using a fluorescent spectrophotometer (Cary Eclipse, Agilent Technologies) at excitation wavelength of 351 nm (λex). The FL spectra were collected by addition of 0.5 mL of solutions including Ca2+ ions (known and unknown concentrations) into the 2 mL of SA-4CO2Na aqueous solution (0.5 mM). 3
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swelling environment (Supplementary Fig. S3), was observed during both swelling and degradation (Fig. 2a). Interestingly, while the magnitude of SD rates during swelling (from t = 0 to 30 min) and early stage of degradation (from t = 30 to 60 min) were approximately the same ( ± 0.067%/min), a constant release rate of Ca2+ (0.046 mM/ min) was observed within the range of 0 to 60 min. When the SD rate reduced to −0.0023%/min (60 < t < 120 min), an obvious decrease in the release rate of Ca2+ became apparent (0.0034 mM/min). Similar behaviour indicating the change in FL intensity (Supplementary Fig. S4), weight and release of Ca2+ ions was observed when the crosslinking time increased to 5 min, as shown in Fig. 2b. During 240 min of swelling process (Region II in Figs. 2b and 3), the rate of water uptake was different at various time intervals, i.e., 0.015, 0.008 and 0.003%/ min for 60, 120 and 240 min of swelling, respectively. Similar trends for the release rate of Ca2+ were found identifying 0.067, 0.025 and 0.007 mM/min release rates of Ca2+ in the swelling intervals of 60, 120 and 240 min, respectively. From 240 to 360 min, an equilibrium region was appeared (Region III in Figs. 2b and 3), where the change in FL intensity, weight and Ca2+ ion release were negligible [17,19]. The ionic-crosslinking is one of the most common methods to prepare hydrogels using sodium alginate solutions [35–39]. Using this method, a sodium alginate hydrogel forms by ionotropic gelation using Ca2+ ions via creation of chelate structure, known as egg-box model of cross-linking [34]. Since the chelate structure allows a high degree of coordination of Ca2+ ions, the created junction during crosslinking forms a kinetically stable towards dissociation. On the other hand, the anionic alginate chains are likely to form temporary junctions by reacting with Ca2+ ions showing a normal polyelectrolyte characteristics of cations binding [40] and takes part into the swelling process, as shown in Fig. 3I. During the initial stage of swelling process, the diffusion of water results in dissociation of temporary junctions; hence, the counter ions (Ca2+) expel the hydrogel network resulting in increase of the density of fixed negative charges, as shown in Fig. 3II and the Region II of curves in Fig. 3a. The dissociation of temporary junctions can't degrade the whole hydrogel, however the lattice size of the network would enlarge due to the repulsion of negative charges and the swelling ratio increases, as shown in Fig. 3III. When the lattice size reaches to a critical value the chelate junctions would be disjointed, resulting in degradation of the hydrogel, as shown in Fig. 3IV. The feasibility of the proposed method for monitoring of Ca2+ release during the SD process was further investigated by two common cases, including the impact of the addition Na+ ions, which is a common situation for the relevant studies [40] and application of ultrasound waves during swelling. The swelling behaviour of dried sodium alginate droplets (at 37 °C for 24 h) that were crosslinked for 5 min was studied in distilled water and NaCl (3% w/w) solution (Fig. 4a). The addition of Na+ ions into the swelling environment resulted in decrease of swelling ratio, compared to that of the immersed hydrogel in distilled water. After 200 min of swelling, a significant difference in swelling ratio was found between the samples were placed in distilled water and NaCl solution. The FL intensity (Supplementary Fig. S5) and the amount of released Ca2+ ions (Fig. 4a inset) were lower in the presence of Na+ ions. The increase in concentration of Na+ ions in the swelling environment enhances the ionic strength of the solution (compared to water) and decreases the difference of osmotic pressure inside the hydrogel network to that of the solution. Thus it can be concluded that the Ca2+ ions that are available at the temporary junctions, control the initial stage of the swelling process. Thanks to the insensitivity of SA-4CO2Na to Na+ ions (Supplementary Fig. S6), the read of higher Ca2+ concentration during swelling process was reliable [33]. When the SD process was performed under sonication, a change in FL maxima (Supplementary Fig. S7) was observed compared to the control (without sonication), identifying the high release of Ca2+ due to the impact of sonication process on dissociation of chains at the chelate structure (Region IV in Fig. 3), as demonstrated in Fig. 4b.
Fig. 4. (a) Swelling behaviour of dry sodium alginate hydrogels in distilled water and NaCl solution and the concentration of released Ca2+ monitored by the AIEgen after 200 min (insert) and (b) the release study of Ca2+ from sodium alginate hydrogels measured by the AIE proposed method in the presence of sonication compared to the control and the images of the hydrogel after 25 min (inset).
3. Results and discussion The concentration of SA-4CO2Na was constant (0.5 mM) among all experiments and the FL intensities were measured 30 min after collection of samples to get the stable reading, as shown in Fig. 1a and Supplementary Fig. S1. The relative FL intensity changes of the AIEgen at 560 nm upon addition of different concentrations of Ca2+ ions were prepared, as shown in Fig. 1b and Supplementary Fig. S2 to establish a relationship (as demonstrated by Eq. (2)) between the relative FL intensity and the Ca2+ concentration, which can be used for quantitatively monitoring Ca2+ release during the SD process.
I = 2.443 + 1.338C Ca2 + I0
(2)
where I is the FL intensity at time t and I0 is the initial FL intensity at time = 0. CCa2+ represents the concentration of Ca2+ at time t. Different SD behaviour was observed for hydrogels that were prepared at different crosslinking times of 1 and 5 min. An increase in crosslinking time resulted in distinction between SD rates and appearance of a plateau region (Fig. 2b) before initiation of degradation (Fig. 2a and b). For short crosslinking time (t = 1 min), a continuous increase in the FL intensities reflecting the release of Ca2+ into the 4
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While the results presented here are convincing that AIE-based method is more compelling compared to the gravimetric techniques, one limitation is the specificity towards Ca2+ ions. Therefore, suggested approach exclusively can be used for hydrogels that are crosslinked by Ca2+ ions including alginate base hydrogels [41–44], gellan [45] and xanthan [46] gums. However, our recently published study revealed that AIE-based techniques are able to accurately measure the swelling properties of different hydrogels [47].
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
AIEgen quantitatively monitoring the release of Ca2+ during swelling and degradation process in alginate hydrogels
T
Javad Tavakolia, Esther Laisaka, Meng Gaob, Youhong Tanga,
⁎
a
Institute for Nanoscale Science and Technology, Medical Device and Research Institute, College of Science and Engineering, Flinders University, South Australia 5042, Australia b National Engineering Research Center for Tissue Restoration and Reconstruction, School of Material Science and Engineering, South China University of Technology, Guangzhou 510006, China
ARTICLE INFO
ABSTRACT
Keywords: Alginate hydrogels Aggregation-induced emission (AIE) Calcium release Swelling Degradation
Alginate-based hydrogels are extensively used for different biomedical applications. While the swelling and degradation of alginate-based hydrogels affect their structure-property relationship, many studies employed gravimetric analysis to characterize the swelling-degradation process. Accurate or not, this traditional method is difficult to be consistently performed with minimized errors, especially at the late stage of the process. For the first time, this study introduced a reliable, accurate and cost-effective method to minimize the human-sourced errors during repetitive measurement of swelling and degradation of alginate-based hydrogels based on Ca2+ specified aggregation-induced emission fluorogen technology. This study provides an approach for characterization of different properties of alginate-based tissue engineered scaffolds. The established relation between the changes in released Ca2+ into the swelling environment and its relative intensity identified the potential application of the proposed method for prediction of swelling and degradation behaviour in alginate-based hydrogels.
1. Introduction Recent evolution in the design of new biomaterials has been focused on naturally-driven materials that are able to mimic the function of extracellular matrix of tissues and regulate the host responses [1–6]. Owing to the inherent biocompatibility, alginate is a natural material with wide range of applications in medical fields [7,8]. Alginate is a water-soluble anionic polymer that extracted from brown algae, i.e., seaweed by treatment with alkali solution [9]. From a structural point of view, alginate is a polysaccharide copolymer of (1-4)-β-D-mannuronic acid and (1-4)-α- L-guluronic acid, where their ratio depends on its natural source [10]. When crosslinked with divalent cations, for example Ca2+, a biocompatible alginate hydrogel with structural similarity to the extracellular matrix of living tissues is formed that allows wide applications in tissue engineering [11], delivery of bioactive agents and macromolecular pharmaceutics [12], cell transplantation [13] and wound healing [14,15]. The swelling and degradation (SD) of alginate-based hydrogels that affect their biomedical applications can be controlled by partial oxidation using sodium periodate, change in crosslinking duration (time) or concentration and regulating the molecular weight distribution
⁎
[16,17]. Since the biocompatibility of alginate hydrogel is likely to decrease at high degrees of oxidation [17], the use of low molecular weight sodium alginate and optimized concentration of crosslinking agent is a proper approach that offers control over the SD rates. No matter which of the strategies are used, the most frequent practical approaches to characterize the SD rates are gravimetric methods, measuring the weight change in hydrogel, as shown in Eq. (1) or analysing the viscosity of swelling environment over time [18].
SD Changes =
Mt
M0 M0
× 100
(1)
where, M0 and Mt are weights at the initial and time t, respectively. Moreover, several studies employed the DSC analysis and mechanical properties measurement to indirectly address the SD process, since a change in glass transition temperature and ultimate strength of hydrogel is expected [18,19]. Accurate or not, the traditional methods are difficult to be consistently performed with minimized errors, especially at the late stage of SD process. Since the fluorescence (FL) properties of aggregation-induced emission fluorogens (AIEgens) is improved by the restriction of intramolecular rotation (RIR) in aggregated state, they have been widely
Corresponding author. E-mail address: [email protected] (Y. Tang).
https://doi.org/10.1016/j.msec.2019.109951 Received 26 November 2018; Received in revised form 4 June 2019; Accepted 5 July 2019 Available online 07 July 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 2. Change in the weight and release of Ca2+ ions during SD process of sodium alginate hydrogels crosslinked for (a) 1 and (b) 5 min. The vertical red dot lines identify the swelling (region II), equilibrium (region III) and degradation (region IV) phases. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. (a) Dynamics of relative FL intensity change with SA-4CO2Na (0.5 mM) in CaCl2 solution (100 mM), inset: Chemical structure of SA-4CO2Na and (b) the relative FL intensity change as the function of Ca2+ concentration with 0.5 mM of SA-4CO2Na.
the FL intensity of the collected swelling environment was measured by addition of the AIEgen, simultaneously.
used for fabrication of advanced biomaterials, cell imaging, drug delivery and photodynamic therapies [20–26]. The characters of high emission efficiency in the aggregated state, excellent photo-stability, and high sensitivity make AIEgens suitable for biomedical applications. Currently, the application of AIEgens in tissue engineering and regenerative medicines has become a hot topic since provides a simple framework for better understanding of the interaction between host tissue and biomaterials, resulting in design of more efficient implants [27]. Based on the recent progress reporting wide range of applications for AIEgens [28–32]; here, a new approach for real time monitoring of release of Ca2+ during the SD process of sodium alginate-based hydrogels is presented. A recent study identified that the AIEgen, SA4CO2Na can be well dispersed in an aqueous solution with a very weak fluorescence emission. However, the AIEgen could form highly emissive aggregates in the presence of Ca2+ through electrostatic and chelating interactions [33]. In the current study, this AIEgen, SA-4CO2Na, was used for real-time detection and quantification of Ca2+ ions that are released during the progression of SD process. To demonstrate this concept, low viscosity sodium alginate and 100 mM CaCl2 solutions were used for preparation of droplet alginate hydrogels, as described before [34]. The SD properties of sodium alginate hydrogels were determined by measuring the changes in hydrogel's weight. Meanwhile,
2. Materials and method 2.1. Materials and reagents Sodium alginate (Alginic acid sodium salt from brown algae, Low viscosity, A1112) and CaCl2 (anhydrous, granular, C1016) were purchased from Sigma-Aldrich. The AIEgen, SA-4CO2Na, was a gift from our collaborator laboratory at Hong Kong University of Science and Technology, Hong Kong, China. 2.2. Sample preparation A stock solution of SA-4CO2Na in distilled water with a concentration of 50 mM was prepared and then diluted to 0.5 mM solution and kept in a sealed glass bottle. The solution was wrapped in aluminium foil and stored in a refrigerator under 4 °C for further use. Sodium alginate (4% w/w), NaCl (3% w/w) and CaCl2 (100 mM) aqueous solutions were prepared by mixing appropriate amount of powder in distilled water at room temperature and stirring (using magnet stirrer) for 5 h and 5 min, respectively. A 3 mL syringe (gauge 18) was used for preparation of sodium alginate hydrogel beads by injection of sodium alginate solution (4% w/w) into the CaCl2 solution (100 mM) under 2
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Fig. 3. Schematic drawings of SD process in sodium alginate hydrogels (I) early stage of swelling, (II) swelling region where the diffusion of water into the hydrogel network results in breakage of temporary junctions (III) repulsion of fixed negative charges (at final stage of swelling and equilibrium states) increases the uptake of water and resulted in enlargement of lattice size and (IV) degradation of hydrogel network proposed by dissociation of chains at the chelate structure. Alginate chains including chelate structures, calcium ions and water molecules denoted by orange-black, green and blue circles, respectively and (a) the corresponding Ca2+ release and weight change during Regions I-IV. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
slow stirring (50 rpm). Beads were extracted from CaCl2 solution after 1 and 5 min of crosslinking and rinsed with distilled water for 1 min.
Briefly, a standard cuvette (3 mL volume) was filled with 2 mL of SA4CO2Na aqueous solution (0.5 mM). A sodium alginate bead was placed in distilled water or NaCl solution. Then samples including 0.5 mL of swelling environment were collected at each time point using a sampler (1000 μL) and injected into a cuvette that was filled with SA-4CO2Na aqueous solution. All cuvettes were allowed to stand for 30 min before FL data collection (Spectra and intensity). To evaluate the effect of sonication on the release of Ca2+ ions from sodium alginate hydrogels, wet beads (crosslinked for 5 min) with approximately similar initial weights (3.6 ± 0.02 mg) were placed in a cuvette including 2 mL of SA-4CO2Na aqueous solution (0.5 mM). A continuous sonication (sweep mode) was applied to the sample for 10 min time intervals using a sonication bath (MRC) at room temperature and the FL intensities were measured, subsequently. All sample preparation and relevant measurements were performed using sealed containers (cuvettes) to avoid evaporation.
2.3. Characterization The SD process was measured using gravimetric method by measuring the weight of beads that were placed in distilled water and NaCl solution at different time points. Both dry (dried for 24 h at room temperature after crosslinking with CaCl2 Solution) and wet beads (immediately used after crosslinking in CaCl2 solution) were used. The SD process for wet samples was performed immediately after preparation of samples (sodium alginate beads). The detection of Ca2+ was conducted using a fluorescent spectrophotometer (Cary Eclipse, Agilent Technologies) at excitation wavelength of 351 nm (λex). The FL spectra were collected by addition of 0.5 mL of solutions including Ca2+ ions (known and unknown concentrations) into the 2 mL of SA-4CO2Na aqueous solution (0.5 mM). 3
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swelling environment (Supplementary Fig. S3), was observed during both swelling and degradation (Fig. 2a). Interestingly, while the magnitude of SD rates during swelling (from t = 0 to 30 min) and early stage of degradation (from t = 30 to 60 min) were approximately the same ( ± 0.067%/min), a constant release rate of Ca2+ (0.046 mM/ min) was observed within the range of 0 to 60 min. When the SD rate reduced to −0.0023%/min (60 < t < 120 min), an obvious decrease in the release rate of Ca2+ became apparent (0.0034 mM/min). Similar behaviour indicating the change in FL intensity (Supplementary Fig. S4), weight and release of Ca2+ ions was observed when the crosslinking time increased to 5 min, as shown in Fig. 2b. During 240 min of swelling process (Region II in Figs. 2b and 3), the rate of water uptake was different at various time intervals, i.e., 0.015, 0.008 and 0.003%/ min for 60, 120 and 240 min of swelling, respectively. Similar trends for the release rate of Ca2+ were found identifying 0.067, 0.025 and 0.007 mM/min release rates of Ca2+ in the swelling intervals of 60, 120 and 240 min, respectively. From 240 to 360 min, an equilibrium region was appeared (Region III in Figs. 2b and 3), where the change in FL intensity, weight and Ca2+ ion release were negligible [17,19]. The ionic-crosslinking is one of the most common methods to prepare hydrogels using sodium alginate solutions [35–39]. Using this method, a sodium alginate hydrogel forms by ionotropic gelation using Ca2+ ions via creation of chelate structure, known as egg-box model of cross-linking [34]. Since the chelate structure allows a high degree of coordination of Ca2+ ions, the created junction during crosslinking forms a kinetically stable towards dissociation. On the other hand, the anionic alginate chains are likely to form temporary junctions by reacting with Ca2+ ions showing a normal polyelectrolyte characteristics of cations binding [40] and takes part into the swelling process, as shown in Fig. 3I. During the initial stage of swelling process, the diffusion of water results in dissociation of temporary junctions; hence, the counter ions (Ca2+) expel the hydrogel network resulting in increase of the density of fixed negative charges, as shown in Fig. 3II and the Region II of curves in Fig. 3a. The dissociation of temporary junctions can't degrade the whole hydrogel, however the lattice size of the network would enlarge due to the repulsion of negative charges and the swelling ratio increases, as shown in Fig. 3III. When the lattice size reaches to a critical value the chelate junctions would be disjointed, resulting in degradation of the hydrogel, as shown in Fig. 3IV. The feasibility of the proposed method for monitoring of Ca2+ release during the SD process was further investigated by two common cases, including the impact of the addition Na+ ions, which is a common situation for the relevant studies [40] and application of ultrasound waves during swelling. The swelling behaviour of dried sodium alginate droplets (at 37 °C for 24 h) that were crosslinked for 5 min was studied in distilled water and NaCl (3% w/w) solution (Fig. 4a). The addition of Na+ ions into the swelling environment resulted in decrease of swelling ratio, compared to that of the immersed hydrogel in distilled water. After 200 min of swelling, a significant difference in swelling ratio was found between the samples were placed in distilled water and NaCl solution. The FL intensity (Supplementary Fig. S5) and the amount of released Ca2+ ions (Fig. 4a inset) were lower in the presence of Na+ ions. The increase in concentration of Na+ ions in the swelling environment enhances the ionic strength of the solution (compared to water) and decreases the difference of osmotic pressure inside the hydrogel network to that of the solution. Thus it can be concluded that the Ca2+ ions that are available at the temporary junctions, control the initial stage of the swelling process. Thanks to the insensitivity of SA-4CO2Na to Na+ ions (Supplementary Fig. S6), the read of higher Ca2+ concentration during swelling process was reliable [33]. When the SD process was performed under sonication, a change in FL maxima (Supplementary Fig. S7) was observed compared to the control (without sonication), identifying the high release of Ca2+ due to the impact of sonication process on dissociation of chains at the chelate structure (Region IV in Fig. 3), as demonstrated in Fig. 4b.
Fig. 4. (a) Swelling behaviour of dry sodium alginate hydrogels in distilled water and NaCl solution and the concentration of released Ca2+ monitored by the AIEgen after 200 min (insert) and (b) the release study of Ca2+ from sodium alginate hydrogels measured by the AIE proposed method in the presence of sonication compared to the control and the images of the hydrogel after 25 min (inset).
3. Results and discussion The concentration of SA-4CO2Na was constant (0.5 mM) among all experiments and the FL intensities were measured 30 min after collection of samples to get the stable reading, as shown in Fig. 1a and Supplementary Fig. S1. The relative FL intensity changes of the AIEgen at 560 nm upon addition of different concentrations of Ca2+ ions were prepared, as shown in Fig. 1b and Supplementary Fig. S2 to establish a relationship (as demonstrated by Eq. (2)) between the relative FL intensity and the Ca2+ concentration, which can be used for quantitatively monitoring Ca2+ release during the SD process.
I = 2.443 + 1.338C Ca2 + I0
(2)
where I is the FL intensity at time t and I0 is the initial FL intensity at time = 0. CCa2+ represents the concentration of Ca2+ at time t. Different SD behaviour was observed for hydrogels that were prepared at different crosslinking times of 1 and 5 min. An increase in crosslinking time resulted in distinction between SD rates and appearance of a plateau region (Fig. 2b) before initiation of degradation (Fig. 2a and b). For short crosslinking time (t = 1 min), a continuous increase in the FL intensities reflecting the release of Ca2+ into the 4
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While the results presented here are convincing that AIE-based method is more compelling compared to the gravimetric techniques, one limitation is the specificity towards Ca2+ ions. Therefore, suggested approach exclusively can be used for hydrogels that are crosslinked by Ca2+ ions including alginate base hydrogels [41–44], gellan [45] and xanthan [46] gums. However, our recently published study revealed that AIE-based techniques are able to accurately measure the swelling properties of different hydrogels [47].
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