Thermo/Shear-Responsive Injectable Hydrogels from an Alginate/PNIPAM-Based Graft Copolymer: Effect of Divalent Cations Ca 2+

: This work is focused on the design and development of biocompatible self-assembling hydrogels, which behave as soft gels at room temperature and strong gels at the physiological temperature, suitable for potential bio-applications. A graft copolymer of sodium-alginate, bearing eight side chains of poly(N-isopropylacrylamide), enriched with the hydrophobic comonomer N -tertiary-butyl-acrylamide (NtBAM), (NaALG-g-P(NIPAM-co-NtBAM)) were used as gelator. In to-tal, 5 wt% aqueous polymer solutions in the presence of Ca 2+ cations were prepared and evaluated as thermo-responsive hydrogels. Rheological experiments revealed a twostep reversible gelation either upon heating or cooling. The divalent cations operated as a cross-linking agent through ionic interactions, inducing the formation of a network at low temperatures. Upon heating, an additional crosslinking develops through thermo-induced hydrophobic association of the thermo-responsive P(NIPAM-co-NtBAM) side chains above a critical temperature. The combination of thermo- and shear-responsiveness provides shelf-assembling systems as potential candidates for injectable strategies. For instance, the system under investigation could be used for cell transplantation, which requires a weak gel to protect the cells during injection and a gel strengthening after the injection at a physiological temperature to immobilize the created scaffold in the targeting position of the host tissue.


Introduction
Hydrogels are constituted of three-dimensional networks, highly swollen in aqueous media [1]. Hydrogels derived from natural polysaccharides have attracted great interest in recent decades due to various potential applications in biomedical science, arising from their properties, such as biocompatibility, non-toxicity, and biodegradability. The gelation capabilities of the polysaccharide macromolecules can be tuned using their functional groups through grafting strategies. For instance, polysaccharides grafted with associative pendant chains (stickers) can form 3D networks with reversible crosslinks [2]. A very interesting category of hydrogels, capable of responding to external stimuli, e.g., temperature and pH, etc., are called "smart" hydrogels [3]. Provided that the network can be formed upon responding to a stimulus, injectable hydrogels can be designed, rendering them potential candidates for drug and cell delivery systems.
Alginate hydrogels retain a structural similarity to the extracellular matrices in tissues, and, as a result, these gels have promising applications in biomedicine and tissue engineering. Alginate is a natural and linear polysaccharide obtained from brown algae, consisting of (1-4) linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) units [4]. Alginic acids as negatively charged polymers exhibit the ability of gel formation via ionic interactions with divalent cations, such as Ca 2+ and Mg 2+ etc. [5]. The most common used cation is Ca 2+ . The junctions between Ca 2+ and the carboxy groups of alginates are described as the egg-box model [6].
Thermo-responsive alginate-based graft copolymers were developed recently. The hydrophilic backbone of alginate is grafted by the commonly used thermo-responsive polymer of N-isopropylacrilamide (PNIPAM), which exhibits a lower critical solution temperature (LCST) at about 32 °C, below the physiological solution. This critical temperature is referred to as the high molecular mass PNIPAM [7]. Hence, upon the heating procedure, hydrophobic associations of the thermo-responsive stickers occur above a critical gelation temperature (Tgel) and beyond a percolation concentration, leading to a self-assembling network in water [3]. The reversible behavior operates upon cooling. More importantly, the LCST can be tuned by enriching the thermo-responsive homopolymer chains with a comonomer [8]. The addition of a hydrophobic comonomer decreases the LCST to lower values and in turn the sol-to-gel transition temperature. This effect also influences all the rheological properties and can be used to tune them at 37 °C (physiological conditions).
The aim of this work was to explore the behavior of a thermo-responsive alginatebased hydrogel in the presence of divalent cations Ca 2+ . For this purpose, we used an alginate as a gelator, grafted by eight thermo-responsive side chains of poly(N-isopropylacrylamide), enriched with the hydrophobic comonomer N-tertiary-butyl-acrylamide (NtBAM). The main interest of the present work was to endow the thermo-responsive system with combined properties by adding Ca 2+ ions as an additional cross-linking agent. Through the ionic interactions between the cations and the anions along the Na-Alginate backbone of the gelator, a soft gel forms at a lower temperature. Upon heating, additional hydrophobic association of the side chains occurs. Overall, the system exhibits a soft to strong gel transition below and above the physiological temperature.

Synthesis of the Graft Copolymer NaALG-g-P(NIPAM94-co-NtBAM6)-NH2
The grafting "onto" methodology was used to accomplish the synthesis of alginatebased graft copolymer. Briefly, -NH2 end-functionalized P(NIPAM94-co-NtBAM6) random copolymers were grafted onto the -COO − groups of sodium alginate (NaALG) through carbodiimide chemistry. The resulting copolymer bear eight thermo-responsive grafting chains. The molar ratio of the NIPAM/NtBAM monomers in the P(NIPAM94-co-NtBAM6) side chains was 94/6 (mol/mol) and the Mn was 14,800 g/mol. The overall weight composition of the graft copolymer was 53 wt% NaAlg and 47 wt% P(NIPAM94-co-NtBAM6). Details of the synthesis and characterization are reported elsewhere [8].

Hydrogels Preparation
To produce solution with 4 mM concentration of Ca 2+ , 0.0059 g of calcium chloride dihydrate (CaCl2 H2O) was dissolved into 10 mL distilled water and left stirring under room temperature for 24 h. This solution was used as the aqueous media. Aqueous solutions of sodium alginate graft copolymer were prepared at a concentration of 5 wt%. After homogeneity, the pH of the solutions was regulated at the philological value of 7.4, using NaOH (1 M).

Rheological Studies
A stress-controlled AR-2000ex (TA Instruments) rheometer with a cone and plate geometry (diameter 20 mm, angle 3°, truncation 111 μm) was used to investigate the rheological properties of the sodium alginate graft copolymer aqueous solutions in terms of shear-and thermo-response. The experiments were accomplished in the linear viscoelastic regime, determined by strain sweep tests at a frequency of 1 Hz. The samples were loaded at a Peltier plate system that ensured the experimental temperature with high accuracy (±0.1 °C). The rheometer was equipped with a solvent trap to prevent water evaporation during the experiments.

Results
To explore the thermo-induced properties of the NaALG-g-P(NIPAM94co-NtBAM6) aqueous solution in the presence of Ca 2+ ions, rheological measurements were carried out through a temperature ramp oscillatory shear experiment. A heating/cooling cycle was accomplished with a rate of 1 °C/min. As shown in Figure 1a, the elastic modulus G' predominates the loss modulus G'' in the entire temperature region, denoting the formation of a 3D network. At low temperatures, below the LCST of the side chains, the network formation was ascribed to the intermolecular ionic interactions arisen from the presence of Ca 2+ ions (egg-box model). Upon heating, and above a critical temperature (at about 30 °C), the moduli increased significantly and a stronger network formed due to the intermolecular hydrophobic association of the grafting side chains, as an addition to the Ca 2+ crosslinking. Importantly, both phenomena were reversible. In Figure 1b, tanδ is presented as a function of temperature. In all cases, tanδ was lower than 1, confirming gelation. Moreover, the hydrogel strengthened with the increase of temperature, while tanδ decreased steadily with temperature. We observed that the gel strengthening was more pronounced above the critical temperature, due to the additional gelation arisen from the thermo-induced side chain association.  in Figure 2. As can be observed, the storage modulus was higher than the loss modulus in the frequency range investigated, and the terminal relaxation zone was not visible in all investigated temperatures, implying the formation of a 3D network. Moreover, the moduli increased with temperature in agreement with the temperature ramp data. The injectability of the hydrogel was evaluated in terms of shear-and thermo-responsiveness, simulating experimental conditions similar to those of an injection through a 28gauge syringe needle as depicted in Figure 3. By switching the shear rate from 0.01 s −1 to 17.25 s −1 at 20 °C (injection at room temperature), a remarkable shear-thinning effect was observed, as the viscosity decreased instantaneously by about two orders of magnitude. Upon decreasing the shear rate at 0.01 s −1 and simultaneously increasing the temperature at 37 °C (after injection at body temperature), the viscosity was instantaneously raised by three orders of magnitude. The viscosity was then higher than that at 20 °C, with more than one order of magnitude conforming to the thermo-response of the system. By repeating the experiment, the system showed excellent responsiveness and reversibility. linear viscoelastic regime. At high strains, G'' becomes higher than the G', as demonstrated in Figure 4a, implying the destruction of the network. At Figure 4b, a time sweep experiment was conducted with a strain value within the linear regime and the temperature at 37 °C. As seen, the network (hydrogel) recovered almost instantaneously, since the storage modulus prevailed the loss one and at higher magnitude due to thermo-response. The initial retardation was due to the temperature equilibration process of the rheometer from 20 to 37 °C.

Conclusions
A 5 wt% aqueous polymer solution of a graft copolymer of sodium-alginate, bearing eight P(NIPAM94-co-NtBAM6) thermo-responsive side chains, were investigated in the presence of 4 mM Ca 2+ cations. The rheological data revealed a twostep gelation. At lower temperatures, a soft gel formed through ionic interactions between the divalent cation and the carboxyl anions of alginate. Upon heating, a secondary hydrophobic crosslinking of the thermo-responsive side chains occurred, leading to strong gel. Overall, the presence of Ca 2+ transformed the behavior of the system from a sol-to-gel transition (without Ca 2+ ) to a soft-to-strong gel transition (with Ca 2+ ). The prepared hydrogel exhibited excellent injectability and self-healing, induced by shear and temperature. These thermo-and shear-responsive shelf-assembling networks could be potential candidates for injectable strategies for stem cell transplantation This process requires a weak gel to protect the cells during injection and a stronger gel after injection to immobilize the created scaffold in the targeting position of the host tissue.

Conflicts of Interest:
The authors declare no conflict of interest.