**3. Magnetic-responsive hydrogels**

Hydrogels with magnetic responsiveness have also recently received great attention to developing the next generation of stimuli-responsive hydrogels that possess unique functional structures with controllability, actuation, and spatiotemporal response properties controlled by an external magnetic field. Such magneto-responsive hydrogels have also been used in a multitude of applications in the biomedical field, such as enhancement of cell growth and differentiation for tissue regeneration, drug delivery controlled by magnetic fields, magnetic hyperthermia for the treatment of cancer or magnetic actuators [55]. Magnetic nanomaterials are composed of magnetic elements (e.g., Fe, Co, and Ni) and their oxides (e.g., Fe3O4, Fe2O3, and CoFe2O4) [56], and although widely investigated in terms of their physical, structural, and magnetic properties, little is still known about their full potential impact on the biomedical field. Among those nanomaterials, magnetite (Fe3O4) has become the most used for medical applications not only because of its biocompatibility and non-cytotoxicity but also for its tunable magnetic properties [55]. Thus, the size of the NP has an effect on the induced magnetic moment and the magnetic properties (e.g., ferromagnetic, superparamagnetic, etc.), which in turn can be used to control their orientation and accumulation, or aggregation, within the hydrogel. For example, NP aggregation affects the biological fate of the magnetic NP that prevents their internalization into the cells and therefore their further excretion, increasing their cytotoxicity. Therefore, it is essential to control the chemical (e.g., composition) and physical (e.g., size, shape, etc.) characteristics of magnetite NP since they impact the former properties. For more details, see reference [55].

As with the electrically conductive hydrogels, magnetic nanocomposite hydrogels can also be fabricated by following the same strategies—(i) blending, (ii) in situ precipitation, and (iii) covalent bonding. Again, blending methodology has been widely employed for its simplicity since the polymer and the magnetic nanomaterials are physically mixed followed by the polymer chains crosslinking to get the hydrogel network. Sapir et al. successfully developed a magneto-responsive hydrogel by properly dispersing magnetite NP by sonication in an Alg solution and followed by crosslinking with Ca2+ ions. The magnetic NP, ranging from 5 to 20 nm, did not seem to have any significant effect on the physicochemical properties of the hydrogel, such as porosity, stability, and wetting, as the NP were perfectly embedded within the polymer network but the mechanical and magnetic properties were improved. However, some NP aggregation was observed [57]. Fuhrer and coworkers developed a more complex magnetic hydrogel formed by the incorporation of 4-vinylbiphenyl functionalized carbon-cobalt core-shell NP into an aqueous solution containing 2-hydroxy-ethyl-methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), and styrene-maleic anhydride (SMA). A rheology additive (tetramethyl

ethylenediamine) and a crosslinker (APS) were added and the reaction took place in a casting mold for 1 h at room temperature (**Figure 3A**). Although the magnetic properties of the nanomagnets were not reported, the authors did observe an influence of an external magnetic field on cell differentiation [58].

In situ precipitation was employed as a method to avoid NP aggregation since first a mixture of the metal salts (e.g., FeCl2 and FeCl3), precursors of Fe3O4, were better mixed within the polymeric solution before cross-linking. After that, formation of magnetite was achieved by precipitating the Fe2+ and Fe3+ ions with NaOH following the reaction:

$$\mathrm{Fe}^{2+} + 2\mathrm{Fe}^{3+} + 8\mathrm{OH}^- \rightarrow \mathrm{Fe}\_3\mathrm{O}\_4 + 4\mathrm{H}\_2\mathrm{O} \tag{1}$$

Albertsson et al. fabricated a hemicellulose magnetic hydrogel by a one-step method. First, O-acetyl-galactoglucomannan and epichlorohydrin (crosslinking agent) were dissolved in NaOH followed by the addition of an aqueous solution of the metal salts (FeCl3·6H2O, FeCl2 (Fe3+:Fe2+ molar ratio = 2:1)). Different concentrations were added to incorporate variable amounts of magnetite (5, 10, and 15%). The crosslinking reaction and the magnetite formation simultaneously took place at 60°C for 20 min. The resultant hydrogel contained Fe3O4 NP with an average size of 5.8 nm conferring a superparamagnetic behavior to the hydrogel. Moreover, it was observed that the higher the Fe3O4 content, the higher the magnetization of the hydrogel. The presence of the magnetic NP also improved the mechanical properties but a decreased swelling ratio, thermal stability, and pore size was observed as magnetite content increased [62]. Another example by Miyazaki et al. was the in situ incorporation of Fe3O4 NP within chitosan hydrogels with different crosslinking degrees achieved changing molar ratios of the crosslinker (glutaraldehyde) from 0.5 to 30 with respect to the amino groups in chitosan. The authors immersed the hydrogels with different crosslinking densities in a 0.1 M FeCl2 solution for 6 h at room temperature to allow hydrogel swelling and Fe2+ diffusion into it. After that, hydrogels were dipped into 0.5 M NaOH solution at 60° to precipitate the magnetic NP. An influence of the hydrogel network structure on magnetite growth was observed. On one hand, the amount of Fe3O4 generated in the hydrogel decreased as the crosslinking density increased, which they attributed to the lower swelling meaning that the Fe2+ intake was impeded. On the other hand, larger crystallite sizes were obtained as the crosslinking degree was increased. The authors did not show the magnetic properties of the hydrogels but they did analyze the influence of an alternating magnetic field in heat generation for hyperthermia applications. The heat generation was enhanced in hydrogels with higher crosslinking due to the larger crystallite and particle sizes and despite the lower amount of magnetite [63]. Zhou et al. also fabricated hydrogels based on PVA or PVA/PNIPAAM and containing Fe3O4 NP as the magnetic material. The one-step process consisted of mixing a PVA or PVA/PNIPAAM solution with the Fe2+/Fe3+ ions followed by dropwise addition into an alkaline NH3 solution to obtain the magnetite NP and crosslink the PVA chains in the form of beads (**Figure 3B**). PVA played different roles, the stabilizer to avoid magnetite aggregation and the matrix to support the NP. On the other hand, the Fe3O4 NP interacted with the hydroxyl groups of PVA via hydrogen bonds favoring also gelation. Although the authors did not provide the size of the magnetite NP, they did report their superparamagnetic behavior indicating a nanometric size. They finally incorporated congo red inside the magnetic scaffold to be used as a drug delivery system. They found out a different profile release with and without an applied magnetic field [59].

#### **Figure 3.**

*(a) (I) Photo of the magnetic hydrogel with a dog-bone shape. (II) TEM image of the carbon-coated metal nanomagnets. (III) TEM image of the nanomaterials incorporated into the hydrogel. Reproduced with permission from Ref. [58]. (b) (I) Images of magnetic PVA hydrogels in the form of beads. (II) Magnetization-magnetic field curves for the hydrogels with different magnetic contents at 300 K. Adapted with permission from Ref. [59]. Copyright (2012) American Chemical Society. (c) (I) Scheme showing the synthesis of CoFe2O4 NP coated with citric acid (CA) (CoFe2O4@CA) and 3-methacryloxypropyltrimethoxysilane (MTS) and the corresponding hydrogels MBA-FHG and NP-FHG. TEM images of the (II) CoFe2O4 NP coated with and (III) the swollen and freeze-dried magnetic hydrogel. Adapted with permission from Ref. [60]. Copyright (2011) American Chemical Society. (d) Scheme showing the experimental procedure to align the magnetic particles and collagen fibres: (a) Liquid collagen suspension with neurons (orange) and magnetic NP (red). (b) Placement of the suspension onto coverslips and allowed to solidify with (bottom) or without (top) magnetic field. (c) Final scheme of the random and aligned hydrogels. SEM image of (II) the random distribution of magnetic NPs within collagen hydrogel and (III) the magnetic strings in the hydrogel solidified under a magnetic field. Adapted with permission from Ref. [61]. Copyright (2016) American Chemical Society.*

#### *Hybrid Hydrogels with Stimuli-Responsive Properties to Electric and Magnetic Fields DOI: http://dx.doi.org/10.5772/intechopen.102436*

A third strategy to incorporate magnetic NP has been covalent bonding, which implies the formation of a covalent bond between the functionalized magnetic nanomaterials and the polymer chains. Although this method usually involves more complicated steps and is more time-consuming, the advantage is the prevention of NP leaching out from the hydrogel network. For example, PAA and methacrylic surface-functionalized CoFe2O4 NP were employed to assure a covalent bonding between them. First, the CoFe2O4 NP were synthesized by precipitation from a CoCl2 and FeCl3 (1:2 molar ratio) solution after alkalinization and stabilized with citric acid and tetramethylammonium hydroxide in water. And second, functionalization was obtained after mixing the NP first with NH4OH solution (25%) and second with 3-methacryloxypropyltrimethoxysilane (MTS) allowing the reaction (e.g., condensation of siloxane groups onto particle surface) to take place at room temperature for 15 h. The resultant particles were single-crystalline and had an average size of 12.2 ± 0.23 nm, resulting in a pseudo-superparamagnetic behavior. Finally, the authors synthesized different PAA hydrogels with the citric acid- and methacrylicfunctionalized CoFe2O4 NP to investigate the effect of the particle-to-polymer interaction (hydrogen bonding in citric acid- and covalent bonding in methacrylicfunctionalized particles) on the hydrogel magnetic and mechanical properties [64]. Another example was the formation of covalently bonding hydrogels between siloxane-functionalized CoFe2O4 NP and the PVA matrix (**Figure 3C**). The procedure was very simple as they first mixed the monomer (AAM) and the functionalized CoFe2O4 NP followed by the crosslinker (N,N,N′,N′-tetramethylethylenediamine) and the initiator (ammonium peroxodisulfate). The reaction proceeded at room temperature for 2 h. The magnetic NP had a size around 12 nm showing a superparamagnetic behavior. Moreover, they also showed that the hydrogel swelling was lower when the NP were covalently bonded to the hydrogel compared to the NP physical entrapped [60].

In all previous strategies, a more or less homogeneous but random distribution of the magnetic NP can be achieved. Recently, the development of complex hydrogels architectures has grown by controlling the spatial distribution and orientation of the magnetic nanomaterials within the hydrogel scaffold by using an external magnetic field. Normally, the magnetic nanomaterials are mixed with the hydrogel precursors and subsequently aligned by placing the mixture in an external magnetic field. The nanomaterials become magnetized and reorient along the magnetic field direction. This anisotropic and well-ordered structure is then fixed by crosslinking the precursor solution into the hydrogel. Although this approach can be applied to magnetic nanomaterials with different shapes (e.g., NP, NR, and NW), magnetic NP have been extensively used for the preparation of such anisotropic hydrogels [61, 65]. For example, Fe3O4 NP have been successfully aligned within the hydrogel precursor solution containing AM (monomer), MBAM (crosslinker), APS (initiator), and tetraethylethylenediamine (accelerator) using a static magnetic field. After that, the hydrogel formation was triggered by increasing temperature up to 50°C. Such alignment led to an enhanced magnetothermal effect under an external alternating magnetic field compared to the disordered hydrogel [66]. Or Antman-Passig and Shefi embedded Fe3O4 NP in a collagen fiber suspension, aligned the NP into strings under an external magnetic field, which also forced the alignment of the fibers, and finally, collagen was allowed to solidify keeping the magnetic field (**Figure 3E**). The seeded neurons had normal electrical activity and viability and their growth was induced and controlled along the fibers and NP string direction acting as a physical cue for the cells [61].

#### **3.1 Biomedical applications**

These magnetically-responsive hydrogels have enabled a wide range of potential applications in the biomedical field, such as tissue engineering, drug delivery, artificial muscles, soft actuators, and magnetic hyperthermia, among others. Tissue engineering has been one of the fields where magnetic hydrogels have been widely applied covering a wide range of tissues, such as bone, cartilage, cardiovascular, or neuronal tissues. The ultimate aim of scaffolds is to foster the natural reparative process by guiding the new tissue formation and recovering their functionality, where multiple biochemical, biophysical, and biological cues need to be controlled. Magnetic hydrogels are key in this discipline since hydrogel architectures can be magnetically controlled in a way to confer directionality and or concentration gradient mimicking complex anisotropic tissues. Moreover, these hydrogels can be remotely actuated with external magnetic fields inducing mechanical deformation within the scaffold (e.g., magnetomechanical stimulation or mechanotransduction effect), which has an impact on cell behavior (e.g., growth, migration, proliferation, and differentiation). For example, Huang et al. have reported an effective regeneration of cartilage using magnetic hydrogels composed of PVA, hydroxyapatite particles, and maghemite (Fe2O3) NP. The incorporation of the NP improved not only the mechanical properties of the hydrogel but also induced the proliferation and differentiation of the seeded MSC into the chondrogenic lineage [67]. Other approaches have applied either static or time-varying magnetic fields to the cells-containing hydrogels. Thus, Brady et al. developed a three-layer agarose-Fe3O4 hydrogel with a stiffness gradient that was achieved using a different agarose concentration in each layer (1, 2, and 3 wt.%). Bovine chondrocytes were embedded in each layer under the application of a 500 mT static magnetic field (**Figure 4A**). After 14 days of magnetic stimulation, they observed an increase in both strain and sulphated glycosaminoglycan content from the 1 wt.% agarose layer to the 3 wt.% agarose layer [68]. Fuhrer and collaborators also observed that the application of a non-continuous (2 s on, 10–225 s off) 800 mT magnetic field to the Fe3O4-styrene-maleic anhydride hydrogel seeded with MSC induced their chondrogenic differentiation without the need of any other chondrogenesis transcription factors [58].

More recently, magnetic hydrogels with anisotropic architectures have been fabricated trying to mimic native tissues. For example, an anisotropic collagen-agarose bilayer containing Fe3O4 NP was obtained when a 2 mT magnetic field was applied during hydrogel formation. Collagen fibers aligned as a consequence of the NP alignment parallel to the field direction but only in the layer where the agarose content was lower (0.5 w/v%). The layer with the higher agarose concentration (1 w/v%) hindered collagen and magnetite NP alignment. The authors observed that seeded chondrocytes in the anisotropic scaffolds expressed more collagen type II when compared with the isotropic hydrogels [72]. These anisotropic structures were also recently explored by Araújo-Custódio et al. who reported the fabrication of gelatin hydrogels containing rod-shaped cellulose nanocrystals decorated with magnetite NP that were aligned by applying a static magnetic field (108 mT). The hydrogel, that tried to mimic tendon tissue, showed a directional structure with anisotropic mechanical properties being the storage modulus higher in the direction parallel to the rod long axis. This anisotropy also had an impact on the embedded cells as it induced an elongated morphology and a directional growth again on the rod long axis (**Figure 4B**) [69].

Another application where magnetic nanocomposite hydrogels have been investigated is drug delivery due to the possibility to release the drug on demand *Hybrid Hydrogels with Stimuli-Responsive Properties to Electric and Magnetic Fields DOI: http://dx.doi.org/10.5772/intechopen.102436*

#### **Figure 4.**

*(a) Live/dead stain images (dead cells (red), live cells (green)) showing the cell viability of chondrocytes embedded into the different layers of the agarose-Fe3O4 NP hydrogel at different times (I) day 1, (II) day 7 and (III) day 14. Reproduced with permission from Ref. [68]. (b) (I) SEM images of isotropic and anisotropic hydrogels. Scale bar = 10 μm. (II) Confocal microscope images showing the effect of isotropic and anisotropic hydrogels on cell alignment (red, cytoskeleton; blue, nucleus). Adapted with permission from Ref. [69]. Copyright (2019) American Chemical Society. (c) (I) Graph showing the amount of congo red loaded onto the hydrogels with different magnetic contents with time. (II) Graph showing the release profiles of the hydrogels with different magnetic contents over time with and without applied magnetic field. Adapted with permission from Ref. [59]. Copyright (2012) American Chemical Society. (d) (I) Photo of the Alg/PEDOT/Fe3O4 NP hydrogel. (II) Graph showing the variation of temperature of the Alg/PEDOT/Fe3O4 NP hydrogel with time subjected to an alternating magnetic field (200 Hz, 8 kA m−1). Adapted with permission from Ref. [70]. Copyright (2021) American Chemical Society. (E) (I) Image of the hydrogel without (left) and with an applied magnetic field (right). (II) SEM images of the hydrogel in the undeformed and deformed states. Scale bar: 500 μm. Reproduced with permission from Ref. [71].*

and at certain concentrations when magnetic fields are applied. Moreover, the delivery of the therapeutic drug in situ to the specific target can be done remotely. In this line, Mahdavinia and collaborators fabricated a magnetic IPN hydrogel network containing k-carrageenan and PVA as well as FeSO4 and FeCl3 to precipitate Fe3O4 NP by adding NH3. After that, diclofenac sodium as a model drug was added to the previous mixture and further crosslinked by the freezing-thawing method followed by immersion in K<sup>+</sup> solution. The hydrogel, that showed a superparamagnetic behavior with magnetization saturation values between 3.4 and 8.2 emu/g depending on the magnetite content, was subjected to an alternate magnetic field with variable strength in the range 100–500 G. They observed a controlled diffusion of the drug such that the higher the magnetic field was, the higher the amount of diclofenac sodium released. They attributed this behavior to the higher mechanical stress conferred as the magnetic field increased [73]. Another example by Zhou et al. showed that the amount of congo red loaded onto the hydrogels was the same independently of the amount of magnetite inside the PVA hydrogel. However, they did observe a change in the amount of congo red released in the absence and presence of a static magnetic field. After 500 min, the released content was around 55% with no magnetic field and around 42% with the applied magnetic field for the hydrogel with the lowest amount of magnetite (**Figure 4C**) [59].

Some other applications of magnetic hydrogels are magnetic hyperthermia as experimental cancer therapy or soft actuators to develop artificial muscles. Magnetic hyperthermia, which basically consists of the delivery of heat when a high frequency oscillating magnetic field is applied, has been investigated into magnetic nanocomposite hydrogels. For example, Puiggalí-Jou et al. observed that when an alternating magnetic field (frequency = 200 kHz) was applied to a Alg/ PEDOT/Fe3O4 NP hydrogel, the temperature increased from room temperature to around 50°C after a few minutes, which was attributed to the presence of the magnetic NP (**Figure 4D**) [70]. More recently, hydrogels with ordered structures like the one fabricated by aligning magnetite NP with a PAM hydrogel have also shown magnetothermal effect but direction-dependent. When the magnetic NP chains were aligned parallel to the applied field, the heating rate and the plateau temperature were higher than the values achieved with the non-ordered hydrogels [66]. Magnetically responsive hydrogels can also be used as soft actuators due to the change in volume, shape, or position they experience in response to a magnetic field. Thus, Zhao et al. developed an Alg hydrogel crosslinked with adipic acid dihydrazide (AAD) and containing magnetite NP with a diameter around 10 nm. The application of a magnetic field (38 A/cm<sup>2</sup> ) induced deformation of the hydrogel with a volume change of 70% (**Figure 4E**) [71]. Zhou et al. developed the amphiphilic pentablock copolymer PAA-PC5MA-PEO-PC5MA-PAA (PC5MA: poly(5 cholesteryloxypentyl methacrylate), PEO: poly(ethylene oxide)) and the Fe3O4 NP that were directly bonded to the carboxylic groups of PAA. These magnetic hydrogels were bent under the application of a magnetic field [74]. Recently, significant efforts have been put into developing dual electric- and magnetic-responsive hydrogels with even enhanced properties compared to the single stimuli-responsive systems. For example, Liu et al. fabricated a flexible hydrogel containing CNT, PPy NP, and iron oxide with electrical conductivity and magnetic properties with potential applicability as biosensor and bioactuator [75]. Or Garcia-Torres and collaborators synthesized an Alg/PEDOT/Fe3O4 hydrogel for magnetic hyperthermia application and simultaneous measurement of temperature [70].

*Hybrid Hydrogels with Stimuli-Responsive Properties to Electric and Magnetic Fields DOI: http://dx.doi.org/10.5772/intechopen.102436*
