Magneto-Responsive Hydrogels: Preparation, Characterization, Biotechnological and Environmental Applications

Hydrogels are hydrophilic three-dimensional networks able to hold large amount of water and hydrophilic molecules. Magneto-responsive hydrogels comprise of magnetic nanoparticles dispersed in polymeric networks that can be manipulated under external magnetic field. This review aims at (i) giving a brief overview on the evolution of hydrogels until the design and development of magnetic hydrogels; (ii) describing the types of hydrogels and the basic concepts about superparamagnetic iron oxide nanoparticles, as well as the preparation and characterization of magneto-responsive hydrogels; (iii) displaying the relevant applications of magneto-responsive hydrogels for drug delivery, regenerative medicine of tissues, cancer therapy and environmental issues and (iv) highlighting the challenges and future trends of magneto-responsive hydrogels for 3D and 4D printing.


Introduction
The paper is organized as follows.The "Introduction" section shows (i) the chronological evolution from gels to magnetic hydrogels; (ii) the classification of polymer hydrogels regarding their chemical and physical aspects and (iii) the basic concepts about superparamagnetic particles."Methods for the Preparation of Magnetic Hydrogels" section presents the different methods for the preparation and characterization of magnetic hydrogels."Biomedical Applications of Magnetic Hydrogel" section describes the application of magnetic hydrogels for drug delivery, tissue engineering and cancer therapy."Environmental Applications" section shows the application of magnetic hydrogels for remediation of contaminated water.Finally, "Future Trends and Challenges" section discusses about the challenges and future trends regarding the 3D and 4D printing of magnetic hydrogels.

A brief overview
At the beginning of this review, it seems relevant to revisit the origins of the "gel" concept.In 1861, Thomas Graham 1 defined "the colloidal condition" of matter, which regarded real solutions of high molar mass substances such as gelatin, albumin, dextran, which could form superior gelatinous hydrates.Approximately 50 years later, Freundlich, Ostwald and Weimarn proposed that colloid could be any substance in a dispersed "colloidal" state, regardless of the molar mass; thus, suspensions of fine inorganic or metal particles or emulsions could also be considered colloids. 2Although the definition of colloids was established, gels were easier to recognize than to define. 3Three points characterized gels: (i) they are coherent colloid disperse systems of at least two components; (ii) they display mechanical properties characteristic of the solid state and (iii) both the dispersed component and the continuous medium extent themselves continuously throughout the whole system.Moreover, there were two fundamental conditions to create gel from solutions: (i) the solid substance should separate from the solution in a finely dispersed "colloidal" state and (ii) the separated solid particles should not deposit at the bottom nor remain as individual moving particles, but they should form a coherent framework throughout the volume. 4It is amazing that despite the simple instruments or low technology available at that time, the fundamental aspects that defined gels are still valid nowadays.One century ago, gels that could be liquefied and solidified by changing the temperature, such as gelatin in water or agar-agar in water, were classified as heat-reversible and non-heat-reversible gels; today we refer to gels with similar behavior as thermoresponsive gels.
Most gels investigated in the remote past were in fact hydrogels, because they were proteins (albumin, gelatin) or polysaccharides (starch, agar-agar) that formed networks in water.In 1960, the term hydrogels appeared in connection with applications where the gels would be in contact with living tissues and the requirements to make them suitable (swelling, porosity, mechanical properties), particularly the crosslinking of 2-hydroxyethyl methacrylate to create hydrogels contact lens. 5Since then, hydrogels have been widely explored for drug delivery systems, biomedical devices and tissue engineering.Natural macromolecules, polysaccharides and proteins proved to be good candidates to compose the hydrogels because they can be easily crosslinked, they are biocompatible and biodegradable.Hydrophilic synthetic polymers, such as poly(ethylene glycol) or poly(vinyl alcohol), and biodegradable poly(lactic acid) are also widely used as hydrogels due to their biocompatibility.
Reversible contraction and expansion of poly(acrylic acid) hydrogels by changing of medium pH were reported by Kuhn 6 in 1949, who glimpsed the similarity to muscle contraction.He proposed that, in water or alkaline medium, the polymer chains were negatively charged, causing electrostatic repulsion among them and gel stretching; under acid medium, carboxylate groups were protonated, allowing the polymer chains to coil and contract.This work by Kuhn practically launched the concept of smart gels.Nowadays, smart or stimuli-responsive hydrogels are defined as those that reversibly undergo dimensional changes in response to environmental stimuli. 7The physical stimuli include changes in temperature or pressure, intermittent exposition to electric field, magnetic field or light.The chemical stimuli refer to changes in the medium pH and/or ionic strength or to the activity of enzymes or to specific molecular recognition. 8articularly regarding the magneto-responsive hydrogels, the first report dates back 30 years ago with the pioneer work by Kost et al. 9 in 1987, who reported about the magnetically stimulated in vivo release of insulin from magnetic poly(ethylene-co-vinyl acetate) hydrogels under oscillating external magnetic field (EMF).Since then, innumerous magnetic hydrogels have been developed for drug delivery, 10 tissue engineering, 11 diagnostics 12 and for environmental issues. 13The latest trends related to hydrogels refer to injectable hydrogels and 3D (bio) printing, which enable precision in the regenerative process due to the precise construction of biomimetic matrices and positioning of cells and biomolecules embedded in the hydrogels. 14The 4D printing concept relies on the condition that the 3D printed object is stimuli responsive, 15,16 making magnetic hydrogels potential candidates for 4D printing because they offer the possibility to undergo reversible shape change under EMF.

Classification of polymer hydrogels
Polymer hydrogels can be made of natural or synthetic polymers, charged or uncharged.Homopolymers (one type of monomeric unit), copolymers (two or more different monomers) and blends of polymers can be used to produce hydrogels.The interchain interactions can be of physical or chemical nature. 17In physical gels, the polymer chains are kept together by hydrogen bonds, hydrophobic or van der Waals forces and chemical hydrogels contain covalent bonds between polymer chains. 18Changes in temperature, pressure or mechanical stress might affect the stability of physical gels.Contrarily, chemical hydrogels present high stability upon changes in the physical and chemical conditions because the polymer chains are chemically crosslinked. 19Based on the pore size, hydrogels can be classified as nanogels (1 to 100 nm), 20 microgels (100 nm to 5 µm) 21 and macrogels (> 1 mm).All these features, schematically represented in Figure 1, drive the final properties and applications of hydrogels.
Interpenetrating polymer network (IPN) is a sub-category of polymer hydrogels and can be classified as full-IPN or semi-IPN. 22For example, considering polymers A and B, in the full IPN the chains from polymer A are crosslinked with each other and chains from polymer B are also crosslinked with each other, but crosslinking among chains from polymers A and B is not present.In the semi-IPN the chains from polymer A are crosslinked with each other and chains from polymer B are only physically entrapped therein.Figure 2 represents chemical and physical hydrogels, semi-and full-IPN.

Superparamagnetic iron oxide nanoparticles
Superparamagnetic iron oxide nanoparticles (SPIONs) are magnetic nanoparticles (MNP), such as magnetite (Fe 3 O 4 ) and maghemite (γ-Fe 2 O 3 ), with superparamagnetic properties. 23Due to their small size (< 50 nm) SPIONs are considered as single magnetic domain and its magnetization is envisaged as the sum of all the individual magnetic moments of each atom that compose the nanoparticle.SPIONs present magnetic anisotropy, which means that the magnetic moment has two stable antiparallel orientations separated by an energy barrier.The magnetization can flip and reverse the direction of magnetic moment as result of thermal fluctuations in a characteristic relaxation time (Néel relaxation time).In the absence of external magnetic field, the magnetization of MNP is in average zero because the time used to measure the magnetization is much longer than the relaxation time.Figure 3A represents a typical behavior of permanent magnets (or large paramagnetic particles), where the magnetization (B, in tesla) is measured as a function of an applied EMF (H, in A m -1 ).A hysteretic behavior is clearly observed because some domains remain aligned in relation to the remanence; the positions (a) and (d) represent the magnetization saturation, (b) and (e) stand for remanence, (c) and (f) correspond to the coercivity in two opposite directions of applied EMF.In the case of SPIONs, represented in Figure 3B, as the EMF increases,  the magnetic domains become aligned until achieving magnetic saturation moment.Upon decreasing the EMF value, the magnetization decreases, presenting no hysteresis or no coercivity.This is a typical behavior of SPIONs, 24 which makes them attractive for diagnostics because in the absence of EMF there is no magnetization.
Considering the relaxation time as an exponential function of the grain volume, the probability of magnetic moment flipping decreases dramatically with the increase of particle size.When MNP are exposed to electromagnetic field with alternated direction, the magnetization flip dissipates thermal energy to the environment, causing the so-called magnetic hyperthermia, which has been largely used as medical therapy to damage malignant cells. 25,26he MNP might be injected directly into the tumor via a catheter and then heat is induced by alternating EMF.][29] MRI is a powerful non-invasive tool that associates the magnetic properties of hydrogen atoms present in the organism with an EMF and a transverse radio frequency pulse to produce images of human body, detection of tumor cells 30 and localization of tissue-engineered implants. 31PIONs with a medium size between 10 and 300 nm are clinically approved. 27However, harmful effects are related to iron concentration (maximal concentration of 10 µg mL -1 ), dosing and exposure time, as well as to interactions with proteins, changes in the hydrodynamic diameter of the particles and composition of particles coatings. 32,33heranostic systems are interesting because they combine cancer therapy with diagnose to allow drug delivering and diagnostic imaging at the same time via targeting SPIONs encapsulated in magnetic hydrogels. 34Therefore, SPIONs together with hydrogels highlight as potential materials for biomedical applications.The synthesis, protection, functionalization, and applications of MNP for biomedical applications have been comprehensively described elsewhere. 33,35 Methods for the Preparation of Magnetic Hydrogels

Preparation of polymeric hydrogels
There are several ways of synthesis of hydrogels such as physical crosslinking, chemical crosslinking, grafting polymerization, and radiation crosslinking. 36Physical crosslinking methods produce polymer chains weakly bonded, whereas the chemical crosslink requires the covalent attachment of chains by bi-or multifunctional molecules (crosslinkers).In comparison to physical hydrogels, chemical hydrogels have advantages as improved mechanical properties and chemical stability. 37However, unreacted residual crosslinkers should be removed (by rinsing, for instance) and toxic crosslinkers, such as epichlorohydrin, 38 glutaraldehyde 39 or N,N-methylenebisacrylamide 40 should be avoided.

Physical crosslinking
Physical crosslinking stems from hydrogen bonding, hydrophobic interaction, van der Waals forces or ionic interactions among the polymer chains. 41,42Methods such as heating or cooling a polymer solution, maturation or aggregation from heat, 43 stereocomplexation 44 and freeze-thaw cycles may be used to prepare physical gels.Repetitive freeze-thaw processes favor microcrystals formation in the polymer structure, have no toxicity issues and do not generate harmful chemical residues. 45Charged polymers can be crosslinked in presence of multivalent ions of opposite charge by electrostatic interaction; 46 one classical example is the gelation of alginate in the presence of Ca 2+ ions.Gelatin is an example of physical hydrogel from our everyday life formed by hydrogen bonds among the macromolecules; it is stable under low temperature, but at high temperature, the H bonds are disrupted, and the gel structure is lost.

Chemical crosslinking
For the production of chemical hydrogels, the most important methods are radical polymerization, condensation reaction, grafting, high-energy radiation and enzymatic reaction.In chemical crosslinking, polymers chains are crosslinked by bi-or multifunctional molecules, such as boric acid, 47 citric acid, 48 glutaraldehyde, 49 divinyl sulfone, 50 glyoxal 51 and ethylene glycol di-methacrylate. 52Grafting method uses high-energy radiation 53 or chemical agents 54 to initiate the polymerization process.Crosslinking catalyzed by enzymes is interesting because the chemical reactions may take place under mild conditions. 55In radical polymerization, crosslinkers and initiators are added to the polymer solution to produce hydrogels quickly, under mild conditions of temperature and pressure. 56,57

Preparation of magnetic hydrogels
Magnetic hydrogels refer to the incorporation of MNP into the polymeric gel.Iron based MNP can be synthesized by thermal decomposition/reduction, coprecipitation, hydrothermal synthesis, micelle synthesis, and laser pyrolysis. 58Co-precipitation, the simplest and cheapest method, is indicated in equation 1, 59 the pH of aqueous Fe 2+ :Fe 3+ (1:2) salt solutions is adjusted to 10 by the dropwise addition of NH 4 OH, followed by heating (ca.75 °C) for 30 min or sonication at room temperature for 10 min. 60The size, shape, and composition of MNP are influenced by the type of reactants and reaction conditions.Controlling synthetic route conditions and addition of stabilizers can increase the number of monodisperse particles. 58,60 (1)

Blending method
Blending method is advantageous because it is fast, simple and cost-effective.In the blending method, firstly MNP are synthesized, as for instance by equation 1 and then mixed with the polymer solution by mechanical stirring, resulting in physical interactions among polymer chains and MNP.Then, a crosslinker might be added to the system to promote in situ polymer chains crosslinking; the MNP remain physically entrapped into the network [61][62][63][64] (Figure 4a).Alternatively, the synthesis of MNP and crosslinking of polymer chains might be done separately.Then, the hydrogels are immersed in the MNP dispersion to promote physical interaction among them (Figure 4a).After that, the hydrogels are rinsed in order to remove the excess of MNP. 65Keeping the hydrogels immersed into MNP dispersions for long period of time might increase the amount of physically bound MNP.However, for biomedical applications it is important to assure that the MNP are not leached from the hydrogels due to cytotoxicity.For instance, xanthan scaffolds containing less than 1% of magnetite was suitable for biomedical application with appropriated levels of magnetization and absence of cytotoxicity. 66

In situ co-precipitation
In the in situ co-precipitation, hydrogels are immersed in the aqueous solution of Fe 2+ and Fe 3+ ions until achieving swelling equilibrium, 67 as depicted in Figure 4b.Then, hydrogels are soaked in a precipitant medium, such as NaOH 68 and NH 3 .H 2 O, 69 to promote the formation of MNP as indicated in equation 1.It is a simple method and provides high MNP loading into the hydrogels, making them dark and with superparamagnetic properties. 70owever, negatively charged hydrogels can form complexes with Fe 2+ and Fe 3+ ions, decreasing the amount of Fe 2+ and Fe 3+ ions available for the co-precipitation.In addition, the crosslinking among the polymeric chains should be unstable under alkali medium; for instance, ester bonds might undergo hydrolysis at pH > 10. 59 Horst et al. 71 compared two approaches to produce ferrogels, blending and co-precipitation.The results indicated that ferrogels produced by co-precipitation loaded higher amount of MNP than those produced by blending.

Grafting method
3][74] For instance, CoFe 2 O 4 particles modified with aminopropyl silane carry amino groups on the surface, which interact strongly with the matrix of carboxymethyl cellulose; the resulting magnetic hydrogel was efficient for drug release under alternating magnetic field. 75,76Covalent bonds among functionalized MNP and hydrogel network provide advantages such as the decrease of MNP leaching from the gel and the homogeneous distribution of the MNP in matrix. 59,73,76It is worth mentioning that the MNP functionalization step is a costly and complex process.In addition, polymeric network should have active sites to interact with the functional groups on the MNP surface. 59

Characterization of magnetic hydrogels
The characterization of magnetic hydrogels usually comprises the determination of magnetic properties, electron microscopy, swelling degree, mechanical tests, infrared vibrational spectroscopy and rheology.
The superparamagnetic properties of MNP alone or embedded in the hydrogel can be accurately evaluated by a superconducting quantum interference device (SQUID) magnetometer.The magnetization curves yield the magnetic saturation and coercivity, as indicated in Figure 3. Transmission electron microscopy (TEM) is important for the determination of MNP size.As aforementioned, the superparamagnetic properties depend on the size of magnetic particles.Scanning electron microscopy (SEM) is generally used to analyze the hydrogels morphology.First, the hydrogels must be freeze-dried and then coated with a thin layer of conductive material.Upon freezing, the crystallization of ice causes volume expansion, destroying the nanopores.In order to avoid this effect, water can be gradually exchanged by tert-butanol or other alcohol, since it does not expand upon freezing.SEM images can be complemented by energy dispersive X-ray spectroscopy (EDS) for the mapping of iron and iron distribution in the sample.
The swelling degree (SD) of hydrogels is generally determined by gravimetry.The weight of swollen hydrogel divided by the weight of dried hydrogel yields the SD value.The more hydrophilic is the polymeric matrix, the higher is the SD value.The experimental procedure can be very simple, requiring only an analytical balance.The freeze-dried matrix is weighed and immersed in water to swell until the hydrogel volume remains constant.Then, the excess of water is removed by filter paper and the swollen hydrogel is weighed again.High resolution tensiometers allow determining the mass of water uptake by the dried matrix as a function of time.In this case, the dried sample is weighed and placed inside a measuring cylinder with porous bottom, which is connected to the measuring unit.A vessel containing water approaches towards the cylinder until the cylinder touches the liquid surface.At this point, water penetrates into the sample by capillarity, causing steady increase of mass.The increase of mass is recorded automatically as a function of time until achieving a mass value corresponding to saturation "plateau". 48ensile and/or compression tests are important to gain insight about the physical stability of hydrogels under stress.They can be performed for freeze-dried and swollen hydrogels.In the former, the Young (E) modulus is determined from the slope of stress-strain curves; the E values depend on the density (ρ) of dried matrix and on the type of pore, for instance, in the case of porous matrices with open cells, E scales with ρ m , where m vary from 1.5 to 2.0. 77The larger the E value, the stiffer is the matrix.In the latter, the compressive force required to deform the swollen hydrogels by a given extent is evaluated. 78nfrared vibrational spectroscopy is particularly important in the case of chemical hydrogels because the presence of bands in the spectra, which might be assigned to chemical groups belonging to polymer chains and crosslinkers, yields a strong evidence for the chemical crosslinking.Spectra obtained for magnetic hydrogels might present absorption bands in the low wavenumber region (< 800 cm -1 ), which are typical for Fe−O stretching in magnetite, maghemite or hematite. 79heological characterization is particularly interesting for magnetic hydrogels that are designed for injectable formulations or for 3D printing.Extremely low or high viscosity gels are not suitable because they flow very quickly or very slowly, respectively.Ideally, the gel viscosity should be adjusted to provide the suitable flow rate for the desired application.For instance, it is easier to inject a gel through a small gage needle (high strain) if it presents shear thinning behavior. 80Polymer gels which exhibit sol-gel transition at body temperature are also interesting because at room temperature they have low viscosity, facilitating the injection through a small gage needle into animals, and at body temperature, they become gels. 81In the case of extrusion-based 3D printing, first the hydrogel or magnetic hydrogel is loaded into a syringe or reservoir with a relative large cross section area at low shear rate.Then, as the gel approaches the printer nozzle and during printing, the cross-section in the system decreases considerably and the shear rate increases.In this process, pseudoplastic or even thixotropic behavior of hydrogels helps optimizing the shear rate profile in the 3D printing nozzle and printing of smooth and bubble free hydrogels strands. 82As the hydrogel is released from the nozzle and deposited onto a surface, the shear rate becomes practically null.In order to obtain shape fidelity of the printed body, the hydrogels should quickly become stiffer under these conditions.In rheological terms this means that the gels recover their structure and assume a solid-like behavior, which can be characterized by either determining the yield stress (minimum stress to start flowing) or the viscoelastic moduli G' and G".At this point, a given yield stress or a quick recovery of the elastic modulus G' is desirable because it helps keeping the printed shape during the hydrogels solidification.Particularly in the case of magnetic hydrogels, the increase in the content of MNP causes the increase of G' mainly due to the formation of large particle cluster. 83

Biomedical Applications of Magnetic Hydrogel
Physical and chemical stimuli include changes in temperature, 84 electric 85 or magnetic field, 86 light, 87 pH 88 and ionic strength. 89Such stimuli can make drug release from responsive hydrogels more sustained.The presence of MNP in hydrogels makes them suitable for magnetically stimulated applications, as depicted in Figure 5.The concept of magnetically stimulated drug delivery was proposed by Langer and co-workers, 9,90,91 where magnetoresponsive hydrogels of ethylene vinyl acetate were loaded with insulin.Since then new magnetic systems have been developed for biomedical applications, as detailed below.

Drug delivery
Responsive hydrogels have been applied as drug delivery systems due to their ability to swell, tunable viscoelastic properties, thermoreversible gelation, porous structure and biocompatibility. 87,92Triggering drug release processes have several advantages over conventional drug administration because they maintain an effective concentration of released drug for prolonged time, minimizing side effects.][95][96] Table 1 presents examples of magnetically stimulated drug release systems.Some of them can be used to the delivery of drugs to the central nervous system due to their capability of being injected into the intrathecal space. 107owever, drug release in brain is yet challenging due to the blood brain barrier. 108Antiparkinsonian drugs such as levodopa and dopamine were entrapped into alginate and xanthan gum in order to evaluate the drug release in vitro and neural cell response.The EMF of 0.4 T stimulated the release of levodopa from magnetic hydrogels, indicating a suitable system to sustained-release of drug. 99Kondaveeti et al. 98 reported dopamine increase from 24 to 33% under the stimulus of EMF.Hydrogel-based hydrophobic drug delivery is carried out by secondary vehicles encapsulated into the hydrogel such as polymeric micelle and surfactants.However, this way can cause low drug loaded amount into polymeric network.Thus, the employment of secondary vehicles is able to overcome issues of hydrophobic drug loaded in the gel and improve the delivery of hydrophobic drug into aqueous medium. 109,110One promising strategy is the delivery of hydrophobic drug in the form of nanoparticles embedded in hyaluronic acid hydrogels 110 or from hybrid beads of alginate, double hydroxides and magnetic graphite nanoparticles. 111The drug release behavior and the porosity of the ferrogels can be affected by turning on and off the magnetic field. 112enerally, in the absence of external magnetic field (off) the drug diffusion to the medium depended mainly on the interactions among drug and carrier.If the interactions among drug molecules and matrix are weak, the drug molecules tend to diffuse rapidly to the medium (burst effect).On the other hand, if the interactions among matrix and drug molecules are strong, the release tends to be slow.In the presence of external magnetic field (on), the magnetic moments of MNP become aligned with the magnetic field rather than randomly oriented.Alternating from on to off mode, induces an oscillatory movement of MNP, causing vibrations in the crosslinked polymer chains, which stimulate the drug release to the medium. 104,113Moreover, MNP can respond resonantly to an alternated magnetic field, producing heat, this is the so-called magnetic hyperthermia.This phenomenon might decrease the interactions between polymer gel and drug, favoring the drug release. 95

Tissue engineering
Tissue engineering and regenerative medicine aim at restoring, maintaining or improving tissue functions.Cell proliferation and differentiation, cell implantation, and delivering of tissue inducing substances, as growth factors (GF), are important issues in this field.Porous 3D biomaterials or scaffolds allow regular transport of gases and nutrients, favoring cell adhesion, growth and differentiation and the regeneration of damaged tissues. 114,115Hydrogels of polysaccharides and/or proteins are attractive to develop scaffolds due to their similarities with the extracellular matrix (ECM), biocompatibility, biodegradability and chemical versatility. 116ybrid hydrogels are those composed of natural and synthetic macromolecules or macromolecules and inorganic particles.They are also promising materials in the development of bio-artificial tissue of corneas, oral mucosa, skin, cartilage and abdominal-wall. 117However, one should note that the viscoelastic modulus of scaffolds plays a crucial role on the cell response.For instance, for osteogenic differentiation, rigid (modulus ca.30 kPa) scaffolds are indicated, whereas for neuronal differentiation soft (modulus ca. 1 kPa) matrices are preferable. 118agneto-responsive hydrogels have been developed to enhance the scaffolds functionalities and to stimulate cellular responses under external magnetic field. 11,59,119For instance, GF can be loaded into the magnetic hydrogels; their delivery to the cell culture medium can be controlled by applying intermittent or continuous magnetic field.Regardless of the method of preparation, the suitability of the magnetic scaffolds for cell proliferation and differentiation must be preliminarily checked, as detailed elsewhere. 120Briefly, (i) the scaffolds integrity should last during the cell culture process; (ii) the scaffolds should resist to the sterilization method (UV radiation, γ-irradiation, ethylene oxide or ethanol 70% v/v) and (iii) the cell viability on the scaffolds should be determined by counting the number of viable cells after 24 and 48 h contact with the scaffolds, in order to evaluate the scaffold cytotoxicity.If the scaffolds are suitable following the aforementioned criteria, the next step is the determination of cell growth curve over one week or longer and cell differentiation, particularly in the case of stem cells.
The evaluation of cytotoxicity is crucial for all biotechnological applications.Patil et al. 121 compiled results about the cytotoxicity of SPIONs for different cells.Most studies indicate that SPIONs exhibit no toxicity at concentration lower than 0.1 mg mL -1 ; 122 in fact, there are commercial products approved by the U. S. Food and Drug Administration, such as Feridex I.V.®, an intravenous injectable formulation of SPIONs and dextran.The toxic effects of SPIONs, both coated and uncoated, stem from their passive diffusion to the cell interior, where they are enzymatically degraded to Fe 2+ ions; the Fe 2+ ions generate reactive oxygen species (ROS) due to redox cycles.ROS can induce oxidative stress in membranes and harm DNA, affecting cell signaling pathways. 123able 2 shows examples of magnetic scaffolds applied for different cell types, in vitro or in vivo, under magnetic stimulation.The experimental results clearly show that regardless of the cell type, static EMF and pulse electromagnetic fields of weak and strong intensities exert beneficial effects on cell behavior, being the increase of cell proliferation the most frequent observation.It is interesting because many of the magnetic hydrogel listed in Table 2 were prepared under different conditions, resulting in scaffolds with different surface energy, stiffness, porosity and surface anisotropy.
Understanding all mechanisms related to the cell behavior under the magnetic stimuli is still challenging due to the complex intracellular signal transduction pathways.However, some mechanisms are well established.For instance, in the case of osteoblasts, the cell-matrix interactions mediated by integrins, bone morphogenetic protein (BMP)-2 gene expression and p38 phosphorylation were substantially activated under static EMF. 127,137Another important issue is related to magneto mechanical effects on the cell-matrix interactions.Under exposition to an external magnetic field, the magnetic poles of MNP tend to orientate to field direction, causing scaffold deformation.If the EMF is alternated, the flipping might cause vibrations and heat.The mechanical movements of small amplitude can open the mechanosensitive ion channels of cell membranes of adhered cells, favoring the Ca 2+ ions influx, an important signaling step; this is the so-called magnetomechanical effect. 138,139The increase of Ca 2+ ions influx in cells cultivated under magnetic stimuli was evidenced by inductively coupled plasma atomic emission spectroscopy (ICP-AES), 60 potentiometric microsensor selective for Ca 2+ ions 64 and calcium fluorescence. 134Interestingly, Tay et al. 134 observed that the Ca 2+ influx did not depend on temperature changes, confirming that hyperthermia alone could not favor ion channels opening.Other hypotheses to explain the increased cell proliferation under magnetic stimuli consider that MNP might have ability to diminish intracellular H 2 O 2 through intrinsic peroxidase-like activity 140 or that MNP can accelerate cell cycle progression, which may be mediated by the free iron (Fe) released from lysosomal degradation. 141n the case of injectable magnetic hydrogels, one might concern about the fate of magnetic hydrogels after injection.Most of the matrices used in the magneto-responsive materials are biodegradable, but the MNP are not.Considering that the polymer matrix undergoes relatively slow degradation, the release of MNP is also expected to be slow; keeping the amount of MNP implanted tissue at low level.After injection of magnetic hydrogels in rats, the MNP were detected in liver and spleen and part of them was excreted. 142n vitro cell culture systems are generally based on multiwall plates or Petri dishes, which are considered 2D substrates.After the desired proliferation/differentiation, the cells are easily removed from the supports.The 2D cell culture has been used not only to study different cell types in vitro but also to design and test new drugs.However, the results observed from 2D cell culture could not accurately represent the rich environment and complex processes observed in vivo.Thus, the 3D cell culture became attractive because it is closer to the microenvironment that cells experience in vivo, particularly the cell-cell and cell-ECM interactions, which are very important for cell signaling. 143he transition from 2D to 3D cell culture is a relevant step forward to the personalized medicine, where the treatment involves tissue regeneration using patients' own cells.There are different approaches for the 3D cell culture; the cells are brought together with ECM and nutrients in rotational/ agitation or hanging drops, 144 microfluidic 145 or magnetic levitation 146 based bioreactors.In the magnetic levitation cells are mixed with magnetic hydrogel and applied EMF using neodymium magnets on the top of the vials; the MNP moved towards the magnets carrying the cells to the liquid/air interface.The levitated cells attracted other cells, favoring cell-cell interactions in solution.

Cancer therapy
Paul Ehrlich proposed the use of "magic bullets" against illness; they should selectively deliver cytotoxic drugs to their designated targets (e.g.cancer cells). 147NP were used as "magic bullets" because they served as agent contrast in the radiation therapy and they could be transported through vascular system under external magnetic field for cancer treatment. 148The first applications of MNP in chemotherapy appeared in the 70's, for example, for the delivery of cytotoxic drugs, as doxorubicin. 149,150][153] Conventional cancer therapy methods might cause side effects and death of healthy cells due to drug release in the off-target sites.Targeting approaches may be achieved by incorporating chemotherapeutics compounds into magnetic nanohydrogels, which can be guided by EMF to the specific tumor site to release the drug therein.For example, a magnetic thermosensitive hydrogel embedded with Bacillus Calmette-Guérin (BCG) was injected into the urinary bladder and guided to the tumor region by magnets for the treatment of bladder cancer. 154In this case, drug delivery under EMF was more efficient than the conventional surgical transurethral resection (TUR), which left about 40% of residual tumor. 155Dox (doxorubicin) was incorporated in chitosan (CS)/dipotassium orthophosphate hydrogels and applied as drug delivery in the osteosarcoma treatment; the results indicated the decrease of side effects in mice compared to Dox administration without carriers. 156The release of Dox/docetaxel from injectable hydrogels by magnetic hyperthermia induced stimuli displayed good biocompatibility, self-healing and injectable properties. 157mong cancer treatments, hyperthermia has been highlighted as a non-toxicity and non-invasive method.Magnetic hyperthermia refers to heating the cancer cells about 43-49 °C by applying alternated EMF. 158There are some advantages in the use of hyperthermia for chemotherapy such as (i) low toxicity; (ii) control of heating conditions; (iii) less invasive; (iv) injectable and (v) limited side effects. 159,160Poly(ethylene glycol)-based magnetic nanohydrogel particles were remotely heated upon exposure to an alternated EMF causing death of glioblastoma cells in vitro. 161The decrease of cancer cell viability was observed for graphene oxide-based hydrogel 162 and chitosan nanofibers. 163Beyond killing of cancer cells, Dox/graphene-oxide/polyethylenimine allowed reducing the side effects on normal tissues. 162able 3 summarizes the aforementioned examples of magnetic hydrogels for cancer therapy.

Environmental Applications
During the last decades, human activities have severely affected the environment.According to the European Environment Agency 173 eight sectors influence the environment mostly namely, energy (production, conversion, and end-use), industry, transport, agriculture, forestry, fishing and aquaculture, tourism and recreation, and households.Particularly, the impact on water sources is a major public concern because life on Earth depends on them.The most common methods to treat contaminated water involve membrane separation, flocculation and coagulation, chemical oxidation, photocatalytic degradation (Fenton's reaction, for instance), and adsorption.Adsorption is simple, efficient, and costeffective.However, the materials and the process used for the adsorbent production should be as green as possible.Materials from renewable sources or biodegradable materials are good candidates.Moreover, the possibility of regeneration and multiple reuses of adsorbents are also very important to create sustainable processes.Operational methods that do not generate byproducts and that require low amount of energy are desirable.Adsorbents designed for specific interaction with some kind of pollutant are particularly attractive, if their synthesis does not generate large amounts of byproducts.For instance, hydrogels containing host molecules such as cyclodextrins 174,175 and calixarenes 176 are interesting due to their hydrophobic cavities, which serve for the inclusion of hydrophobic pollutants.
The concentration of contaminants found in the water sample defines its quality.There are four categories of contaminants: (i) physical contaminants, which are sediments or compounds suspended in water; (ii) chemical contaminants, such as phosphorus compounds, salts of heavy metals, toxins, polycyclic aromatic hydrocarbons, estrogenic compounds and drugs; (iii) microbiological contaminants (viruses, bacteria, parasites) and (iv) radiological contaminants, as for instance, cesium, plutonium and uranium. 177Hydrogels are efficient adsorbents to remove the contaminants or to pre concentrate them, when they are present at a low concentration to be determined by a conventional analytical method. 178enerally, for the adsorption process, the adsorbent can be either packed in a fixed-bed column and the solution containing the contaminant flows through it or the adsorbent is stirred in a tank containing the contaminant.In the former, the formation of bubbles, irregular compaction and slow speed of solution flow and clogging are common problems. 179In the latter, such problems are avoided, but the adsorbent must be separated for recovery.Depending on the size of the adsorbent, sedimentation might be inefficient, and filtration or centrifugation might be costly and time consuming.Magnetic adsorbents offer the advantage of being easily separated from the solution upon the approximation of an external magnet.In comparison with other separation techniques, the magnetically assisted separation is noninvasive, fast, environmentally friendly and low cost (ca.US$ 2.00 per Nd magnet ca.30 mm diameter × 3 mm high).Magnetic hydrogels have been successfully applied for the removal of inorganic and organic contaminants, followed by magnetic separation and recovery, as exemplified in Table 4.In general, after magnetic separation, the adsorbent must be treated with organic solvent (in the case of organic q max values: 11.1 mg g -1 Cu II , 18.9 mg g -1 Cd II , 20.0 mg g -1 Pb II , 15.0 mg g -1 Co II , 13.5 mg g -1 Ni II and 15.5 mg g -1 Cr III 193 Fe 3 O 4 nanoparticles, xylan and poly(acrylic acid) methylene blue q max = 438.6 mg g - 1 194 Carboxymethyl-b-cyclodextrin and Fe 3 O 4 methylene blue q max = 277.8mg g - 1 195 Hydroxypropyl methylcellulose crosslinked with citric acid and EDTA impregnated with Fe 3 O 4 17α-ethinyl estradiol q max = 0.6 mg g -1 , five cycles 196 Graphene oxide foam Fe 3 O 4 Cr VI q max = 258.6 mg g -1 , reduction of Cr VI to Cr III 197 Chitosan and Fe 3 O 4 Acid Red 2 (dye) q max = 90.06mg g - 1 198 CS, b-cyclodextrin and Fe 3 O 4 2-aminopyridine q max = 46.5 mg g -1 , five times 199 PVA: poly(vinyl alcohol); CS: chitosan; PAM: polyacrylamide; EDTA: ethylenediaminetetraacetic acid; CMS-g-PVI: carboxymethyl starch-g-polyvinyl imidazole.
cycles, adjusting the medium pH to a value, which allows desorption without the dissolution of MNP.For instance, rinsing magnetic hydrogel with 5 mol L -1 HCl caused the reduction of 50% in the original adsorption efficiency after five adsorption/desorption cycles, 200 whereas 0.1 mol L -1 HNO 3 led to the complete dissolution of the MNP already after three cycles. 201An alternative for the desorption of metal ions under acid medium is the addition of electrolytes, which have stronger affinity for the matrix than the ionic pollutant.For instance, the desorption of La III from magnetic alginate beads was successfully achieved by eluting with 0.050 mol L -1 CaCl 2 . 188he adsorption efficiency of hydrogels tends to decrease in the presence of magnetite. 189,196This effect can be explained by the reduction of available adsorption sites, since many hydrogels carry hydroxyl and carboxyl groups that interact with Fe−OH groups by H bonding. Thus, the amount of MNP in the hydrogel should be optimized in order to (i) keep the adsorption capacity at an attractive level without leaching to the medium and (ii) provide enough magnetization to enable magnetically assisted separation.Literature reports 202 indicate successful adsorbents with saturation magnetization ranging from ca. 4 to 60 emu g -1 .
One should notice that although magnetic hydrogels are excellent platforms for the removal of pollutants from water, they are not efficient in the separation of oil from water due to the high interfacial tension between oil and water phases.However, for the removal of oil spilt, magnetic hydrophobic 3D structures (foams, sponges, aerogels, cryogels, xerogels) have been prepared and applied successfully. 202

Future Trends and Challenges
3D printing is an additive manufacturing method, which has been explored for different biomedical applications, such as the creation of dental implants or artificial organs or bioprinting of cells.For the manufacturing of tissues and organs there are two main routes: (i) the tissue engineering route, where firstly the scaffold is printed and then it is combined with living cells and GF to create a biomimetic scaffold; and (ii) the direct assembly route, where cells and gel are printed together as 3D scaffolds. 203In situ 3D printing is already a reality in some hospitals 203 and probably will become very popular in the near future because it enables the production of customized implants.Many materials used for the 3D printing of biomedical devices and scaffolds are soft materials, which turn into stiffer materials upon environmental stimulation (light radiation, temperature change, calcium release).The challenges related to this technology include the precise replication of pieces with very fine structures, as for instance, the vascular system, and pieces with surfaces that prevent bacterial adhesion.Another challenge is the change of the implant shape upon stimulation; this is the so-called 4D printing. 15,204D printing requires "smart materials", such as polymers with shape memory, self-healing polymers or composites, which respond timely to a given stimulus.Composites made of MNP and poly(dimethyl siloxane) were recently used for the 4D printing of magnetically responsive threedimensional (3D) structures; for instance, the wings of a butterfly were printed and could flap upon on/off EMF. 205his kind of technology points at the development of novel remotely controlled devices to perform different functions, 206 making the future of magneto responsive hydrogels even more fascinating.

Figure 1 .
Figure 1.Chemical and physical aspects for the classification of polymer hydrogels.

Figure 3 .
Figure 3. Schematic representation of magnetization (B, in tesla) as a function of EMF (H, in A m -1 ) for (A) permanent magnets (hysteretic behavior) and (B) SPIONs (non-hysteric behavior).In (A), the positions (a) and (d) represent the magnetization saturation; (b) and (e) stand for retentivity; (c) and (f) correspond to the coercivity in two opposite directions of applied EMF.

Figure 4 .
Figure 4. Schematic representation of methods to prepare magnetic hydrogels.(a) Blending; (b) in situ co-precipitation and (c) grafting method.MNP are represented by black spheres.Red and white X symbols represent crosslinker and functional groups, respectively.

Figure 5 .
Figure 5. Schematic representation of drug delivery under stimulus of external magnetic field.The drug (red spheres) can be loaded in magnetic hydrogels, which can be shaped as transdermal patches or injected.

Table 3 .
Magneto-responsive materials in cancer therapy ) or with acid solution (in the case of metal ions) to promote desorption and adsorbent recovery.MNP are often resistant to organic solvents, but the contact with acid medium at very low pH can dissolve them.For this reason, the resistance of magnetic hydrogels under acid medium should be evaluated prior to adsorption/desorption pollutants

Table 4 .
Examples of magnetic hydrogels successfully applied for the removal of inorganic and organic contaminants.The number of adsorption/desorption cycles and removal or maximum adsorption capacity (q max ) were indicated for each example Novel sodium alginate supported tetrasodium thiacalix[4]arene tetrasulfonate and Fe 3 O 4 Cu II , Cd II , Pb II , Co II , Ni II and Cr III