Iron(III)-cross-linked alginate hydrogels: a critical review

Abstract

Ionotropic alginate hydrogels are versatile materials for a wide range of applications. Their biocompatibility and biodegradability have made them perfect candidates for biomedical applications such as tissue engineering and drug delivery. The vast majority of the research related to ionotropic alginate hydrogels has been conducted on Ca²⁺-cross-linked alginate. However, alginate can produce hydrogels with a large number of divalent and trivalent cations. In recent years, the cross-linking of alginate with Fe³⁺ cations has attracted increasing interest due to its extraordinary properties. The particular coordination of Fe³⁺ cations has been found to be critical for mechanical strength, porosity, swelling and other physicochemical properties of the material rarely seen in other ionotropic alginate hydrogels. In addition, the rich redox chemistry of Fe³⁺ cations has been exploited for a wide range of applications, such as drug delivery, tissue engineering, or environmental remediation. In this review we highlight the state-of-the-art that concerns Fe³⁺-cross-linked alginate hydrogels, encompassing from properties and synthesis to applications and future perspectives. We believe that this review would stimulate innovative ideas and promote the research of this material, leading to novel functional materials with new and emerging applications.

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Daniel Massana Roquero

Daniel Massana Roquero graduated with Bachelor in Chemistry from Universidad Autónoma de Madrid (Spain) in 2019. After a short research stay at Universiteit Hasselt (Belgium) supported with Erasmus Scholarship, in 2019, Daniel joined groups of Prof. Katz and Prof. Melman at Clarkson University (Potsdam, NY, USA) where he is presently performing PhD study. His scientific interests are in the areas of “smart” signal-responsive materials, particularly based on alginate hydrogels. Daniel's study has resulted in various signal-triggered biomolecule release systems for biomedical and biotechnological applications.

Ali Othman

Ali Othman graduated with MSc in Chemistry from Jordan University of Science & Technology (Jordan) in 2008. Then, in 2019, he received PhD in Chemistry from Clarkson University (Potsdam, NY. USA). Presently, he is a Postdoctoral Research Associate at the Department of Chemistry & Biomolecular Science (Clarkson University). His research interests focus on development of novel functional nanomaterials and their applications for biosensing and environmental remediation, particularly using wearable bioelectronic devices. He co-authored over 20 peer-reviewed papers, 3 patents and 2 book chapters.

Artem Melman

Artem Melman received his PhD in Organic Chemistry from the Weizmann Institute of Science (Rehovot, Israel) in 1997. He was a postdoctoral associate at the University of Oxford (1997-1999), a Lecturer at the Hebrew University of Jerusalem, and a Research Fellow at the National Institute of Diabetes, Digestive & Kidney Diseases (Bethesda, US). Since 2008 he was an Associate Professor at the Department of Chemistry & Biomolecular Science of Clarkson University. He was an author of 75 papers in peer-reviewed international journals (Hirsh-index 24), 3 book chapters, and 11 patents. His research field was bioinorganic and medicinal chemistry, responsive biomaterials, and electrochemically controlled interfaces. Prof. Melman deceased in 2021 and is missed by friends and colleagues.

Evgeny Katz

Evgeny Katz received his PhD in Chemistry from Frumkin Institute of Electrochemistry (Moscow), Russian Academy of Sciences, in 1983. He was a senior researcher in the Institute of Photosynthesis (Pushchino, Moscow Region), Russian Academy of Sciences, in 1983–1991. In 1992–1993 he performed research at München Technische Universität (Germany) as a Humboldt fellow. Later, in 1993–2006, Dr Katz was a Research Associate Professor at the Hebrew University of Jerusalem. From 2006 he is Milton Kerker Chaired Professor at the Department of Chemistry & Biomolecular Science, Clarkson University (NY, USA). He has (co)authored over 500 papers in peer-reviewed journals/books with the total citation over 40 000 (Hirsch-index 93). His scientific interests are in the broad areas of bioelectronics, biosensors, biofuel cells and biocomputing. In 2019 he received international Katsumi Niki Prize for his contribution to bioelectrochemistry. In 2020 Katz was named Fellow of the International Society of Electrochemistry (ISE).

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1. Introduction

Hydrogels are three-dimensional polymeric networks infiltrated with high amounts of water while maintaining their chemical structure.¹ Hydrogels undergo sol–gel transition as a response to chemical or physical stimuli and, by definition, must contain at least 10% of water in their gel state.² Their biocompatibility,³ tunable biodegradability,⁴ physiological stability⁵ and structural and functional diversity⁶ have situated hydrogels as the perfect candidates for biomedical applications⁷ (e.g., drug delivery⁸ and tissue engineering⁹). Recently, advances in hydrogel technologies have unlocked valuable capabilities for a wide range of various novel applications,^10–12 for example, in environmental science,¹³ forensic science,¹⁴ nanotechnology,¹⁵etc. Hydrogels can be produced from synthetic¹⁶ or naturally occurring polymers (biopolymer).¹⁷ Among the ever-increasing number of polymeric matrices that can undergo hydrogel formation, biomaterial-based hydrogels (e.g., alginate,¹⁸ chitosan,¹⁹ cellulose,²⁰ DNA,²¹ polypeptides,²²etc.) raised interest owing to their superior properties, particularly for bio-related applications.

Alginate is a polyanionic heteropolymer extracted from brown algae²³ or some genera of bacteria.²⁴ The biopolymer chain is composed of β-D-mannuronate (M) and α-L-guluronate (G) linked through 1-4-glycosidic bonds (Fig. 1). These G and M moieties are arranged in polyguluronate (GG), polymannuronate (MM), and mixed GM domains distributed along the alginate chains. While alginic acid is not soluble in water, its salts, such as sodium or potassium alginates, can be dissolved forming viscous aqueous solutions. When an alginate aqueous solution finds certain divalent or trivalent cations, it undergoes instantaneous cross-linking forming ionotropic alginate hydrogels.^25,26 Ionotropic alginate hydrogels have been known and employed for decades for in vivo biomedical applications, environmental science, food science, and textile industry, among many other applications.^18,27–34

Fig. 1 Chemical structures of G and M alginate subunits and their arrangement in the biopolymer chain.

The vast majority of applications and studies involving ionotropic alginate hydrogels employed Ca²⁺ as a cross-linking ion. Calcium-cross-linked alginate hydrogels (Ca²⁺–Alg) are well established and are commercially available, for example, as a cell culture matrix. The emphasis in research of Ca²⁺–Alg has left behind other ionotropic hydrogels, such as Fe³⁺–Alg or Ba²⁺–Alg hydrogels. Nevertheless, in the last years, a dramatic increase in the publications involving Fe³⁺–Alg hydrogels has been noticed. Two main reasons can explain this event, being the particular and exclusive chemistry of iron cations (e.g., coordination chemistry,³⁵ photochemistry³⁶) the first. Second, the different cross-linking mechanism of trivalent cations to the alginate polymer has been found to provide novel features compared to those cross-linked by divalent ions. As a result, Fe³⁺–Alg possess stimuli-responsiveness capabilities, redox properties, photosensitivity, enhanced mechanical stability, higher hydrophobicity, and higher absorptivity among other exceptional properties difficult to find in other ionotropic alginate hydrogels and other hydrogels in general. The versatility of the Fe³⁺–Alg hydrogel has been acknowledged recently in the literature finding for it a wide range of applications, including drug delivery, tissue engineering or contaminant removal among others (Fig. 2).

Fig. 2 Overview of the Fe³⁺–Alg hydrogel properties and applications.

The present review pretends to focus and describe, for the first time, all aspects of the research on Fe³⁺-cross-linked alginate hydrogels (Fe³⁺–Alg). This review paper is divided into two main sections. The first section is focused on Fe³⁺–Alg physicochemical characteristics, synthesis and degradation strategies. The second section is dedicated to the state-of-the-art of current applications of this material. To conclude, challenges and perspectives are briefly analyzed.

2. Iron(III)-cross-linked alginate hydrogels – Preparation and Properties

2.1. Cross-linking mechanism

Alginic acid salts, such as sodium alginate (NaAlg), are soluble in water forming viscous solutions. The biopolymer can undergo sol–gel transition in the presence of divalent (e.g., Ca²⁺, Ba²⁺, Pb²⁺) or trivalent (e.g., Fe³⁺, Al³⁺) cations forming ionotropic hydrogels.^{25,26,37–39} The cross-linking by divalent cations can be explained by the so-called egg-box model in which two facing helical stretches of G sequences bind the divalent ion in a chelate type of binding.^40–42 The cross-linking mechanism and structure of trivalent cation complexes with alginate polymer remain under investigation. Fe³⁺ ion is a “hard” metal cation that tends to form complexes with ligands containing oxygen atoms, particularly in negatively charged ligands such as carboxylate groups. Fe³⁺ complexes with polysaccharide ligands (e.g., chitosan,⁴³ λ-carrageenan⁴⁴ and alginate⁴⁵) are very interesting because of their stability and unusual properties. On the other hand, Fe²⁺ ions have “soft” cationic features and bind preferably to neutral ligands containing nitrogen and sulfur atoms. Fe²⁺ ions poorly cross-link polysaccharides and do not convert alginate from its soluble state to a gel. The dramatic difference in the complex formation of Fe²⁺ and Fe³⁺ ions with carboxylate groups, particularly in alginate,^46,47 is evident from comparing stability constants of their citrate complexes which have log K values 3.2 and 11.85, respectively.⁴⁸ Changing the oxidation state of iron cations can result in the reversible sol–gel conversion of alginate (Fig. 3). The limited information regarding the gelation process of Fe³⁺–Alg arises from experimental complications associated with ferric ions, which produce insoluble hydroxides in water at neutral-basic pH values,⁴⁹ competing with the complex formation with alginate. Two gelation models have been proposed for the alginate cross-linking with Fe³⁺ ions. One model suggests that Fe³⁺ is coordinated by alginate resulting in spatially separated Fe³⁺ centers along with polysaccharides^50,51 and particularly in alginate hydrogel.⁴⁵ On the other hand, the colloidal model suggests that Fe³⁺ ions produce oxyhydroxide (FeOOH) colloids stabilized by polysaccharide chains preventing aggregation of the colloidal species and keeping them in the hydrogel matrix.⁵² Nevertheless, the literature found agreement in the following characteristics of Fe³⁺–Alg cross-linking: (i) Fe³⁺ ions bind alginate stronger than most of other trivalent and divalent cations,^53,54 (ii) Fe³⁺ ions have a coordination number of 6 in the Fe³⁺–Alg complex,⁵⁵ and (iii) Fe³⁺ has the capacity to bind not only with G blocks (as Ca²⁺ does) but also with MG blocks of alginate.⁵⁶ The different binding mechanism of Fe³⁺ ions compared to divalent ions such as Ca²⁺ has endowed Fe³⁺–Alg with enhanced physicochemical properties as it will be discussed in the next section. The fraction of carboxylic groups that can bind the Ca²⁺ cation depends mostly on the amount of GG groups available for cross-linking. Thus, one could expect that the cross-linking of Fe³⁺–Alg depends on the amount of GM and GG groups of the alginate polymer backbone which varies from one source to another. Chemical structures of Fe³⁺–Alg can be found in the literature, but being far from certain and having merely a schematic character.^57,58 The coordination chemistry between Fe³⁺ cations and polysaccharides, including alginate, is still in the study and the outcomes of this research will be essential for a better understanding of Fe³⁺–Alg synthesis, properties, and applications.

Fig. 3 Reversible sol–gel transition of alginate in the presence of Fe²⁺ and Fe³⁺ cations, respectively. Notably, Fe³⁺ cations effectively cross-link alginate resulting in the hydrogel formation, while Fe²⁺ cations are not capable of the hydrogel formation because of much weaker coupling to the alginate polymer. Redox transformation of Fe²⁺/Fe³⁺ cations can change reversibly the alginate from its soluble state to the hydrogel and back (adapted from ref. 47. Copyright 2012 American Chemical Society). On the right, SEM image of the Fe³⁺–Alg surface showing the characteristic brain-like morphology.

2.2. Physicochemical properties

The physicochemical properties of ionotropic alginate hydrogels made them very attractive for many applications.^28–34,59 In particular, biocompatibility and biodegradability have been exploited for tissue engineering⁶⁰ and regenerative medicine.⁶¹ Regardless of the cross-linking ion, alginate hydrogels have physicochemical properties that are strongly dependent on the alginate source, concentration, and the gelation method.^62,63 However, given the different cross-linking mechanisms and the extraordinary chemistry of Fe³⁺ cations, certain physicochemical properties of Fe³⁺–Alg have been found to be exceptional compared to other alginate hydrogels cross-linked with different metal cations (Table 1).

Table 1 Ca²⁺–Alg vs. Fe³⁺–Alg Properties

Ca^2+–Alg	Properties	Fe^3+–Alg
Binds GG groups	Alginate–ion interaction	Binds both GG and GM groups
Egg-box model	Binding structure	Unknown
Two-dimensional	Binding	Three-dimensional
Less	Porosity	More
Faster	Swelling rates	Slower
Softer and more flexible	Mechanical	Stiffer and more rigid
Highly hydrophilic	Surface	Less hydrophilic
None	Magnetic	Paramagnetic

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Upon introduction of gelling ions, the very rapid gel formation typically leads to heterogeneous structures characterized by wide pore size range and inhomogeneous cross-linking density, being higher in the external part than in the core of the gel. Visually, Fe³⁺–Alg hydrogel has a yellowish to brown color (Fig. 3).⁴⁷ Under an electronic microscope, Fe³⁺–Alg hydrogels display a rough surface with interconnected vessels resulting in a “brain-like” morphology (Fig. 3).⁶⁴ Because of the extended 3D-cross-linking with Fe³⁺ ions, a very wide pore size distribution with a large number of macro-pores is characteristic of the Fe³⁺–Alg hydrogels (porosity ranging from a few nanometers to dozens of micrometers).⁶⁵ In the case of divalent cations, the egg-box model showed that divalent cations bind alginate in a planar two-dimensional (2D) manner resulting in less porous morphologies.⁶⁶ The different morphology and porosity of the iron cross-linked alginate hydrogel can explain the different swelling behavior of the Fe³⁺–Alg compared to other ionotropic alginate gels. Alginate hydrogels are known to have fast swelling rates.⁶⁷ The increase in water content causes a decrease in cross-linking density, making the hydrogel softer and eventually triggering its fracture and dissolution. This might be a limiting factor for the hydrogel long-term use in biological fluids.⁶⁸ The Fe³⁺–Alg hydrogels have shown slower swelling rates compared to their divalent analogue gels in both acidic and neutral environments.^69,70 This can be explained by the additional bonds between chains that lead to the formation of a three-dimensional structure. The obtained higher density of crosslinks compared to that of divalent ions induces the decrease of the free volume and the water content. In addition, fast swelling rates are associated as well with the hydrophilicity of the hydrogel. A surface wettability study revealed that Fe³⁺–Alg gels possess a less hydrophilic surface compared to that of divalent ion cross-linked gels since the contact angle measured for Fe³⁺–Alg was approximately 40° while the one of Ca²⁺–Alg was around 10°.⁷¹

Ionotropic alginate hydrogels are known to be relatively rigid but fragile. The capability of Fe³⁺ to bind both GG and GM groups of alginate results in a higher cross-linking density compared to that of Ca²⁺–Alg. Hence a more compact network, endowed with better mechanical properties, such as deformability to compressive and extensional stresses or elastic modulus, can be obtained with Fe³⁺–Alg hydrogels.^69,70,72 Fe³⁺–Alg hydrogels were found to be 30–100% stiffer than Ca²⁺–Alg given the higher cross-linking density with elastic modulus surpassing the 0.2 MPa. The Fe³⁺–Alg hydrogels are all paramagnetic, meaning that the magnetization increases proportionally with increasing the applied magnetic field.⁷² Alkali treatment of the hydrogel resulted in the formation of ferrous nanoparticles that provide the material with superparamagnetic properties.^72,73

Overall, the binding Fe³⁺ to both GG and GM groups of the alginate polymer together with the higher binding affinity of Fe³⁺ with carboxylate-containing molecules has endowed the Fe³⁺–Alg with superior physicochemical properties compared to Ca²⁺–Alg. Since the major limitation of ionotropic alginate hydrogels is the leakage of cross-linking ions that weakens the gel and eventually triggers its dissolution, the enhanced binding endows Fe³⁺–Alg hydrogels with superior stability in particular in biofluids or solutions with high ionic strength. The slowest swelling rates and leakage of Fe³⁺ ions are an indirect confirmation of this superior stability which is indeed a huge improvement for the application of alginate hydrogels in long-term applications.

2.3. Hydrogel synthesis strategies

Several strategies have been developed for the synthesis of ionotropic alginate hydrogel films and particles. Methods for producing alginate hydrogels in macro- and nano-meter scales have been compiled in several reviews.⁷⁴ While these reviews are mainly focused on Ca²⁺–Alg synthesis, most of the reported techniques can be extended to other cross-linking ions by replacing the gelling bath solution (typically containing CaCl₂) for the one with the desired ion (e.g., FeCl₃). This section is meant to provide an introduction for the synthesis of Fe³⁺–Alg based on the literature. These methods are expected to work regardless of the alginate composition. However, given the wide range of alginate sources, the optimized conditions reported should be revised and re-optimized for the specific alginate source and the final intended application.

For the majority of their applications, the Fe³⁺–Alg hydrogels are used in the form of beads. The extrusion-dripping method is the most classic strategy for alginate bead synthesis due to its simplicity. Fe³⁺–Alg beads can be synthesized by drop-wise addition of a NaAlg solution into a Fe³⁺-containing solution. The beads are uniform in size and can be synthesized in a bulk quantity (Fig. 4A).⁷⁵ The formation and properties of Fe³⁺–Alg beads produced from different iron salts have been reported.⁷⁵ The rapid diffusion of Fe³⁺ into the alginate droplet promotes the immediate bead formation resulting in a highly inhomogeneous hydrogel. Low amounts of Fe³⁺ can reach the inner core of the bead that remains soft and liquid, while the external part, with the high Fe³⁺ concentration, has a more rigid structure. The homogeneity of ionotropic alginate gels can be controlled by different parameters.⁷⁶ Low molecular weight alginates and low concentration of a cross-linker ion lead to a highly inhomogeneous hydrogel. On the other hand, higher molecular weight alginates and larger concentrations of gelling ions lead to more homogenous ones. However, an alginate hydrogel formed from high molecular weight are usually undesirable due to their high viscosity. The same latter effect can be achieved if the gelation is done in the presence of non-gelling ions (e.g., Na⁺).⁷⁷ Due to technique limitations, the minimum bead diameter that can be obtained with the drop-casting method is in the range of hundreds of micrometers. To achieve hydrogel particles with smaller diameters (micro- or nano-particles), water in oil emulsions⁷⁸ or microfluidic devices⁷⁹ can be employed.

Fig. 4 Schematic representation of the preparation and visual appearance of: (A) Fe³⁺–Alg beads (adapted from ref. 75. Copyright 2018 Elsevier). (B) Fe³⁺–Alg electrodeposited film (adapted from ref. 127. Copyright 2020 American Chemical Society); (C) homogenous Fe³⁺–Alg film (adapted from ref. 84 with permission). Abbreviation used in the figure: NaAlg–sodium alginate (soluble); Fe³⁺Alg–iron(III)-cross-linked alginate (gel); E – potential applied on the electrode; E⁰ – redox potential of the Fe³⁺/Fe²⁺ system.

Enzymatic reactions can be used to produce Fe³⁺–Alg monodisperse nanoparticles.⁸⁰ Multi-copper oxidase enzyme laccase greatly accelerates the oxidation of Fe²⁺ to Fe³⁺ by dissolved oxygen. If the reaction is conducted in the presence of sodium alginate the reluctant Fe³⁺ cations are instantly trapped by alginate chains near the enzyme molecule producing a Fe³⁺-cross-linked shell around it growing with nearly constant rates for every enzyme molecule. This process also depletes alginate chains from the solution, so, if the concentration of alginate is sufficiently low the reaction produces dispersion of highly homogeneous Fe³⁺ – alginate nanoparticles with their sizes tunable in the 30 to 300 nm range.

The redox properties of Fe²⁺ and Fe³⁺ cations can be exploited to electrodeposit Fe³⁺–Alg onto an electrode surface. The electrodeposition of Fe³⁺–Alg was reported first by our group.^47,81Fig. 4B shows schematically the electrodeposition of an Fe³⁺–Alg film onto an electrode immersed in a solution containing Fe²⁺, NaAlg, and electrolyte (e.g., Na₂SO₄) needed for the electrochemical process, upon application of an oxidative potential. The oxidation of Fe²⁺ to Fe³⁺ at the electrode surface triggers the sol–gel transition of alginate, resulting in a thin Fe³⁺–Alg layer. Note that the Fe²⁺ cations originally present in the solution do not cross-link alginate, while the electrochemically generated Fe³⁺ cations do it. The electrodeposition conditions were optimized for a graphite electrode at 35 mM Fe²⁺, 1.5% (w/v) NaAlg and 0.8 V (vs. Ag|AgCl) for 60 seconds.⁴⁷ It should be noted that the required applied potential might be different for electrodes made of different materials or with chemically modified surfaces. The addition of the Fe²⁺-containing solution (freshly prepared) into the alginate solution should be conducted under vigorous stirring to avoid the formation of Fe²⁺–Alg clumps. The thickness of the layer depends mainly on the electrodeposition time.⁴⁷ The Fe³⁺–Alg layer grows at the electrode surface upon continuing the electrodeposition process. However, with longer times the electron transfer becomes more difficult, obtaining less homogeneity and stability in the external region of the film. The Fe³⁺–Alg film was characterized by cyclic voltammetry (E_1/2 = 0.495 V vs. Ag|AgCl) showing a quasi-reversible redox process associated with the reduction/oxidation of iron cations.^47,81

Highly homogenous alginate films can be accomplished by slowing down the gelation process. For example, the low solubility of CaCO₃ has been employed for the synthesis of more homogenous Ca²⁺–Alg gels.^82,83 As CaCO₃ is getting solubilized at extremely low concentration, Ca²⁺ ions can slowly cross-link alginate resulting in uniform Ca²⁺–Alg gels. Based on the same principle, using low concentrations of Fe³⁺ ions, homogenous Fe³⁺–Alg films were synthesized.^84,85Fig. 4C shows a schematic representation of the film synthesis. The slowly drop-wise addition of an Fe²⁺-containing solution to a NaAlg solution under vigorous stirring produces a homogenous pale yellow solution. To prevent the formation of Fe²⁺–Alg clumps an octagonal magnetic stirrer that allows instantaneous mixing was employed.⁸⁴ The gelation of alginate was limited by the slow oxidation of Fe²⁺ cations by the molecular oxygen present in the air resulting in a more homogenous hydrogel.⁸⁵ The hydrogel solidifies within 6 hours with 35 mM Fe²⁺ ion in the original solution, but it becomes slower if the concentration of Fe²⁺ is decreased.

Thin films of Fe³⁺–Alg can be synthesized easily by the addition of NaAlg solution to an Fe³⁺-containing solution. A commonly employed route for the synthesis of Fe³⁺–Alg films is the solvent casting method.⁸⁶ Before the addition of the cross-linking solution, the water of the alginate solution is removed either by heating or freeze-drying.^64,87 The solvent cast process allows the synthesis of polymeric films without adulteration by heat or plasticizers.⁸⁶

2.4. Hydrogel dissolution/degradation

The presence of enough gelling cations (e.g., Ca²⁺, Fe³⁺) guarantees the sol–gel transition forming an ionotropic alginate hydrogel. The hydrogel is stabilized by electrostatic interactions between the cations and the uronic components of alginate. This interaction is weaker than chemical bonds and hence it is expected that alginate hydrogels are susceptible to ion leakage and eventually, gel dissolution (gel–sol transition). This process is enhanced in the presence of cation chelating agents (e.g., citrate, EDTA, phosphate) and high concentrations of competing ions, such as Na⁺ or K⁺.^30,88 Several attempts have been described in the literature to decrease gelling ion leakage and to stabilize ionotropic alginate gels.^89–92 In addition, ionotropic alginate hydrogels can be degraded in the presence of lyase enzymes^93–96 or certain chemicals, such as sodium periodate.⁹⁷

Ion leakage depends mainly on the binding strength with the alginate backbone. The leakage of Ba²⁺ cations was reported as minimal among other metal cations and the Ba²⁺–Alg hydrogels are the most stable compared to other metal–alginate hydrogels.^25,89 The binding strength of Fe³⁺ to alginate was found to be similar to that of Ba²⁺.⁹⁸ The leakage of Fe³⁺ cations from Fe³⁺–Alg beads has been investigated in different conditions^55,69 being influenced by the surrounding conditions such as ionic strength, pH and temperature. Within 24 hours, 30% of the Fe³⁺ leaked from the Fe³⁺–Alg beads in HCl (pH 1.2), while 15% leaked in PBS (pH 7.4).⁵⁵ Similar results were reported by other research groups.⁶⁹ At low pH, carboxylate groups of the polysaccharide get protonated,⁹⁹ and the electrostatic interaction with Fe³⁺ is no longer effective. Thus, faster diffusion of Fe³⁺ from the Fe³⁺–Alg gels can be expected.

As it will be described in more details later (see Section 3.2), leakage of Fe³⁺ cations and eventual dissolution of the Fe³⁺–Alg hydrogels do not allow their long term applications (e.g., in scaffolds), in which superior hydrogel stability is required. However, for some other applications hydrogel instability will be beneficial (e.g., for drug delivery; Sections 3.1–3.3). In this section the main strategies for Fe³⁺–Alg hydrogel dissolution are presented. The optimized conditions reported may differ depending on the alginate source.

The electrodeposition of Fe³⁺–Alg onto an electrode surface added an additional feature to the ionotropic alginate hydrogels.⁴⁷ The electro-responsive hydrogel can be de-gelled upon application of a reductive potential converting the cross-linking Fe³⁺ cations back to Fe²⁺ cations which cannot keep alginate at the electrode surface, thus resulting in the hydrogel dissolution and removal from the electrode surface. As Fig. 5A shows, the alginate film gets dissolved, stimulated by the electric potential, when the Fe³⁺ cations are reduced to Fe²⁺ cations.¹⁰⁰ The effect of the reductive potential value on the rate of the Fe³⁺–Alg layer dissolution was studied and the applied potential was optimized for different layer thicknesses.⁴⁷ The applied potential of 0.1 V (vs. Ag|AgCl) was enough to reduce partially Fe³⁺ to Fe²⁺, in 30 minutes, but in the same time frame, −1.0 V was enough to reduce all Fe³⁺ cations and dissolve the Fe³⁺–Alg layer completely. The slow charge propagation across the polymer film may inhibit the electro-dissolution. The higher reductive potential applied provides a larger driving force for the redox process facilitating the de-gelation process. The charge propagation gets even more difficult when the Fe³⁺–Alg layer is thicker. The electro-responsive feature is inherent of Fe³⁺–Alg and cannot be found in other ionotropic hydrogels, while some other mechanisms for the electro-response of hydrogels have been reported.¹⁰¹

Fig. 5 Schematic representation of the Fe³⁺–Alg de-gelation mechanism and visual appearance stimulated by: (A) electric potential (adapted from ref. 100. Copyright 2017 Wiley-VCH); (B) chelating ions (adapted from ref. 103. Copyright 2012 Royal Society of Chemistry); (C) hydrogen peroxide (adapted from ref. 106. Copyright 2017 Wiley-VCH).

Fe³⁺-Chelating agents, such as phosphate and citrate ions, bind Fe³⁺ ions stronger than alginate, thus, facilitating the Fe³⁺ ions leakage from the Fe³⁺–Alg hydrogel. This process is accompanied by the diffusion of sodium ions (Na⁺) and water into the hydrogel (Fig. 5B), increasing the osmotic pressure inside the gel.¹⁰² Therefore, the gel swells increasing its size and eventually, when not enough cross-linking ions are present, the gel will collapse (Fig. 5B).¹⁰³ The de-gelation kinetics leading to the hydrogel degradation and dissolution depends on the chelating ion concentration, being faster when the concentrations are higher. Physiological concentrations of ascorbic acid and citrate have been employed to study the de-gelation process of Fe³⁺–Alg beads.¹⁰³

Another Fe³⁺–Alg hydrogel degradation process can proceed in the presence of H₂O₂, as shown in Fig. 5C schematically. The reaction cascade leading to the alginate hydrogel degradation is based on a Fenton-type reaction¹⁰⁴ (the “˙” symbol denotes an unpaired electron):

Fe²⁺ + H₂O₂ → Fe³⁺ + HO˙ + OH⁻

These reactions, catalyzed either by iron cations present in the hydrogel or added externally,¹⁰⁵ produce reactive oxygen species (free radicals HO˙), which react with guluronate and/or mannuronate subunits of alginate chains, resulting in alginate chain degradation and the hydrogel dissolution.¹⁰⁶ This reaction produces free alpha-oxy radicals that reduce Fe³⁺ cations back into Fe²⁺ state which regenerate new radicals, thus continuing the radical chain reaction. The dissolution of Fe³⁺–Alg by H₂O₂ added a new feature in the wide use of alginate as a signal-responsive material. Hydrogen peroxide can be produced in situ in course of many enzymatic reactions (typically catalyzed by oxidases, e.g., glucose oxidase, lactate oxidase, etc.) activated by various biomolecule substrates (e.g., glucose, lactate, etc.). It should be noted that H₂O₂ is present in biofluids (blood, urine, etc.)¹⁰⁷ at small concentrations (1–5 μM) under normal physiological conditions¹⁰⁸ or at elevated concentrations under pathophysiological conditions,¹⁰⁹ both sufficient for oxidative decomposition of Fe³⁺–Alg, thus allowing its use as a drug-delivering matrix with the drug release stimulated by H₂O₂.

Finally, yet importantly, light induced dissolution of Fe³⁺–Alg has been achieved in presence of sacrificial carboxylates.^85,110 The well-known photoreduction of Fe³⁺ to Fe²⁺ in iron carboxylate complexes^36,111,112 can be exploited for dissolution of the Fe³⁺–Alg hydrogels. The photo-responsive behavior of Fe³⁺–Alg hydrogels with different carboxylates has been studied, concluding that compounds containing an α-hydroxyl carboxylic group (e.g., lactic acid) had higher photoreduction rates than those without α-hydroxyl carboxylic group (e.g., butyric acid).⁸⁵

3. Iron(III)-cross-linked alginate hydrogels – applications

The above overviewed physicochemical properties of the Fe³⁺–Alg hydrogels have been exploited in the last decade for a broad range of applications. The rich chemistry of Fe³⁺–Alg hydrogels allowed applications which are not possible for other hydrogels, particularly not achievable with the broadly used Ca²⁺–alginates. The wide versatility of Fe³⁺–Alg hydrogels is gathered and analyzed in this section.

3.1. Drug delivery

An enormous amount of polymeric matrices has been developed and used for drug delivery applications.^113,114 These materials enhanced therapeutic treatments by (i) allowing targeted delivery of the drug; (ii) protecting the drug from degradation, and (iii) preventing undesired immunologic response. Alginate hydrogels (mostly exemplified by Ca²⁺-alginates) have been implemented for delivery systems because of their biocompatibility, ease of synthesis, and high encapsulation efficiency.¹¹⁵ Ionotropic alginate hydrogels have been employed for encapsulation and release of nanoparticles,¹¹⁶ proteins,¹¹⁷ enzymes,¹¹⁸ growth factors,¹¹⁹ small molecules,¹¹⁷ and DNA¹²⁰ among many other bioactive species.¹²¹

The delivery processes of most alginate systems are based on diffusion out of encapsulated species through the alginate pores. Small molecules diffuse faster than larger ones and negatively charged molecules diffuse out of the negatively charged alginate matrix faster than the positively charged ones. The kinetics of the release, so-called conventional release or leakage (Fig. 6), is highly influenced by the surrounding conditions (pH and temperature) and the physicochemical properties of the entrapped (bio)molecules (size, concentration, and isoelectric point). The release time-profile is characterized by an initial burst release that slows down as the gel empties. The uncontrolled release (leakage) of the loaded species is typical for Ca²⁺-cross-linked alginate hydrogels.¹²² The leakage process is obviously controlled by the system composition and its environment, but it is not triggered by any specific signal.

“Smart” drug delivery systems permit a more controllable drug release and have multiple advantages compared to the conventional release (leakage) (Table 2). These materials can encapsulate drug payload and release it at specific targets (targeted delivery) or in specific environments (signal-triggered delivery). The several possibilities to dissolve the Fe³⁺–Alg hydrogel (Section 2.4) make it a multi stimuli-responsive material (light, pH, electric, H₂O₂) with promising applications for signal-triggered and “smart” release of (bio)molecules or drugs.¹²³ This responsiveness, a key characteristic of the Fe³⁺–Alg, cannot be found in other ionotropic alginate hydrogels (cross-linked with other metal cations Ca²⁺, Ba²⁺, etc.) since it is particular of Fe³⁺ ions. The unique properties of the Fe³⁺ cross-linking cations are mostly based on their redox transformation to Fe²⁺ cations, which do not keep alginate in the gel state. When the signal is present in the surroundings of the system, the Fe³⁺–Alg gets dissolved producing a burst release of entrapped (bio)molecules or drugs (Fig. 6). The release is no longer only dependent on the properties of the payload and a similar time-dependent release profile can be expected for any encapsulated molecule in the hydrogel.

Table 2 Advantages of “Smart” (signal-triggered) vs. Conventional Release (leakage)

Conventional release (leakage)	“Smart” (signal-triggered) release
Uncontrolled release profile	Defined drug release profile
Poor drug adsorption	Specific targeting
Premature metabolism/degradation	Drug protection
Side effects	Better patient compliance
Poor drug bioavailability	Enhanced bioavailability

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Fig. 6 Schematic representation of conventional release (leakage) and “smart” signal-triggered release of molecules from Fe³⁺–Alg hydrogel and their time-dependent release profiles.

3.2. Conventional release (leakage)

Delivery of small molecules (e.g., ibuprofen,⁶⁶ folic acid¹²⁴) or proteins, (e.g., BSA frequently used as a model protein⁶⁹) from millimeter-sized Fe³⁺–Alg beads has been analyzed under different conditions. The pK_a of alginic acid is situated around 3.5. The guluronic and mannuronic acid moieties of alginate become protonated at acidic pH <3.5 and the resultant hydrogel is no longer supported by electrostatic interactions, but by hydrogen bonding only (Fig. 7A).¹²⁵ As a result, the hydrogel shrinks leading to a less porous material. For example, the release profile of ibuprofen (Fig. 7B) is very fast (a few hours of time-scale) under neutral pH conditions.⁶⁶ This can be explained by the high porosity of Fe³⁺–Alg hydrogels. However, the release of small molecules was significantly delayed at acidic pH values due to the above-mentioned reason. The same phenomenon was observed for the delivery of proteins, such as BSA (Mw. 66 kDa). However, in this instance, the release of proteins was delayed much more (up to 30 h) due to the larger size of BSA molecules (Fig. 7C).⁶⁹ The conventional release of molecules was highly influenced by the presence of chelating agents such as phosphate (PO₄³⁻). Then, the erosion of Fe³⁺–Alg enhanced remarkably the release rates of proteins and small molecules (Fig. 7B and C).^66,69 In a similar approach, significant increase in the release rates of small molecules of rhodamine 6G was observed in the presence of citrate ions (note that citrate binds Fe³⁺ ions stronger than phosphate ions).¹⁰³ The conventional release (leakage) of DNA from Fe³⁺–Alg films electrodeposited on an electrode surface has been experimentally analyzed and modeled theoretically.^126–128 The release kinetics of DNA was extremely fast due to the presence of negatively charged phosphate groups in DNA resulting in great repulsion within the negatively charged alginate matrix.¹²⁷

Fig. 7 (A) Alginate monomers chemical structures below and above the alginate pK_a value. (B) Release of ibuprofen at pH 2.0 and pH 7.4; the curves correspond to differently prepared samples – see the original publication for details (adapted from ref. 66. Copyright 2018 Elsevier). (C) BSA release in different pH solutions (adapted from ref. 69. Copyright 2015 Wiley-VCH).

3.3. “Smart” (signal-triggered) release

In this section, the signal-triggered release of (bio)molecules (“smart” delivery) from Fe³⁺-Alg is discussed and analyzed.

3.3.1. Electrochemically stimulated release. The electrodeposition and electro-stimulated dissolution of Fe³⁺–Alg hydrogels on an electrode surface have been reported recently.⁴⁷ The electrochemical deposition of the Fe³⁺–Alg film onto a graphite electrode was performed from an aqueous solution containing alginate and Fe²⁺ cations upon application of +0.8 V (vs. Ag|AgCl). Electrochemical oxidation of the Fe²⁺ cations resulted in the production of Fe³⁺ cations near the electrode surface, then rapidly cross-linking the alginate and forming a hydrogel film growing at the electrode surface. When BSA was added to the solution, it was physically entrapped into the growing alginate film. The amount of the entrapped protein was dependent on its concentration in the parent solution and the thickness of the alginate hydrogel film deposited on the electrode surface. Changing the electrode potential to the reductive values (+0.1, −0.4 or −1.0 V vs. Ag|AgCl; all thermodynamically sufficient for the Fe³⁺ reduction) resulted in returning iron cations inside the film to Fe²⁺ cations, which are not capable to cross-link the alginate polymer, then dissolving the hydrogel film and releasing the loaded BSA. The rate of the alginate dissolution and the BSA release process was dependent on the potential applied and it was increased with the reductive potential elevated. Under optimized conditions, the vast majority of BSA was released in less than 30 minutes.⁴⁷ Notably, the electrochemically stimulated release was much faster than the BSA leakage from the alginate film. While the first study included the BSA load and then electrochemically stimulated release, considering BSA as a convenient model, real drugs and other bioactive substances can be released from the Fe³⁺–Alg-modified electrodes in a similar way. In a similar approach, entrapped lysozyme (an antimicrobial enzyme) was electrochemically released and then applied to an agar plate swabbed with Micrococcus luteus (a Gram-positive bacteria) resulting in inhibition of the bacteria growth.⁸¹ It should be noted that the electrical potentials and Fe³⁺ reductive current were applied on the Fe³⁺–Alg-modified electrodes using an external electric device (an electrochemical analyzer).

In a more interesting approach the electrochemical release system can operate as a self-powered device in a way similar to biofuel cells.^129,130 The Fe³⁺–Alg-modified electrode containing pre-loaded biomolecules or drugs operated as a cathode producing current for the Fe³⁺ reduction stimulating the alginate dissolution and the payload release. Its operation was not different from that described above. However, the difference was in the operation of the anode electrically coupled to the releasing electrode. The anode produced the reductive potential and current for the operation of the releasing electrode and the system did not require any external source of electric power for stimulating the release process. In other words, the whole device was self-powered similarly to a biofuel cell. In the first system, the reductive potential was generated at the anode using NADH as electron-donating species (Fig. 8A). NADH was produced enzymatically in various ways, mimicking Boolean logic operations,¹³¹ through reactions catalyzed by different enzymes (Fig. 8B), and then it was electrocatalytically oxidized to form NAD⁺ at a pyrroloquinoline quinone (PQQ)-modified electrode (Fig. 8C). It should be noted that PQQ is an efficient electrocatalyst for oxidation of NADH, particularly in the presence of Ca²⁺ cations in solution.¹³² The reductive potential generated biocatalytically at the anode was transferred to the Fe³⁺–Alg releasing electrode (cathode) connected to the anode with a conducting wire. This system was employed for: (a) the release of an antibody complex (Fig. 8D) that reacted with a complementary-target mimicking a targeted delivery biomedical process;¹³³ (b) the release of β-amylase (β-Amy) and maltose phosphorylase (MPh) (Fig. 8E) that were biocatalytically degrading starch to yield glucose that was further employed in a biofuel cell;¹³⁴ (c) release of a DNA strand that can hybridize with the complementary strand activating a deoxyribozyme (DNAzyme)¹³⁵ utilized in a DNA computing system¹³⁶ (Fig. 8F). In another configuration, a biocatalytic anode was activated for producing the reductive potential/current in the presence of bacterial cells (E. coli)¹³⁷ or antibodies,¹³⁸ then stimulating release processes at the connected Fe³⁺–Alg electrode. The process stimulated in the presence of E. coli cells resulted in the Polymixin B (antibacterial drug) release, then inhibiting the bacteria proliferation.¹³⁷ Importantly, this system realized negative feedback when the bacteria appearance resulted in the bacteria growth inhibition.

Fig. 8 (A) NADH oxidation catalyzed in the presence of PQQ. (B) Enzyme-catalyzed NADH production; three different enzymes were activated with three substrates operating in parallel. (C) Schematic representation of a typical Fe³⁺–Alg electrode used for: (D) release of antibodies (adapted from ref. 103. Copyright 2014 Royal Society of Chemistry); (E) release of enzymes, then producing glucose from starch for biofuel cell operation (adapted from ref. 134. Copyright 2014 Wiley-VCH); and (F) release of DNA, then activating a DNAzyme for DNA computing operation (adapted from ref. 135. Copyright 2015 Wiley-VCH). Abbreviations used in the figure: GDH – glucose dehydrogenase; AlcDH – alcohol dehydrogenase; G6PDH – glucose 6-phosphate dehydrogenase; Glc – glucose; EtOH – ethanol; Glc6P – glucose-6-phosphate; HRP – horseradish peroxidase; β-Amy – β-amylase; MPh – maltose phosphorylase.

The biocatalytic anode used for stimulation of the releasing Fe³⁺–Alg electrode can be activated by different means, using well-established processes typical for various biosensors and biofuel cells.¹³⁹ In one of the possible configurations, the biocatalytic anode was functionalized with PQQ-dependent glucose dehydrogenase (PQQ-GDH) oxidizing glucose (Glc) to gluconic acid (GlcA) and producing the reductive potential/current utilized at the connected Fe³⁺–Alg electrode to release the entrapped Protein A.¹⁴⁰ While glucose present in the solution was used as the electron donor for the anodic process, the anode activation or inhibition (ON/OFF switch) was achieved by pH changes produced in situ by other enzymes mimicking Boolean Not-XOR logic operation. Finally, the release of Protein A from the alginate-electrode was stimulated when the PQQ-GDH-modified anode was in the ON-state controlled through the logic operation. The present system illustrated the possibility of the release function controlled by orthogonal biocatalytic processes switching ON–OFF the biocatalytic reaction at the connected anode. Various biomolecule association/dissociation processes proceeding at the biosensing anode and controlled by pH changes¹⁴¹ or biomolecule signals¹⁴² were utilized to activate the biocatalytic anode and then to trigger the release process from the connected Fe³⁺–Alg-modified electrode. The versatility of the biochemical reactions used for activation of the release process allows construction of various systems for numerous applications. Some of them provide background for future biomedical applications. For example, a system releasing insulin in response to the ketone body (a biomarker of ketoacidosis) was tested in vitro,¹⁴² but it's in vivo operation is potentially possible, at least conceptually.

3.3.2. Release stimulated by H₂O₂. Alginate hydrogel can be disrupted by oxygen-containing free radicals (˙OH and ˙OOH) produced through a Fenton-type reaction of H₂O₂ catalyzed by iron cations (Fe³⁺/Fe²⁺) (see Section 2.4). Then, biomolecule/bioactive species loaded in the Fe³⁺–Alg matrix (e.g., DNA^106,127 or insulin¹⁴³) are released upon alginate decomposition. The H₂O₂ can be directly added to a solution, then defusing into the Fe³⁺–Alg hydrogel resulting in its decomposition and the entrapped species release.¹²⁷ In this case, H₂O₂ is a primary chemical signal activating the release process. Alternatively, H₂O₂ can be produced in situ in reactions catalyzed by enzymes (oxidases)^106,143,144 or by nanozymes (inorganic nanoparticles mimicking enzyme reactions).¹²⁷ Then, the catalytic reactions leading to H₂O₂ production are activated by enzyme substrates serving as primary signals. The reactions can be catalyzed by a single enzyme or nanozyme activated by one substrate (e.g., glucose oxidase, GOx, activated with glucose¹⁴³ or Au-NP nanozyme also activated with glucose¹²⁷) or by several enzymes catalyzing a reaction cascade in the presence of various substrates operating as primary input signals.^106,144 The complex biocatalytic cascades activated by different combinations of the substrate-inputs can mimic various Boolean logic gates.¹⁴⁵ Importantly, glucose serving as a primary input signal can stimulate insulin release, thus, realizing a physiologically meaningful combination of the input signal (glucose) and output result (insulin release).¹⁴³

The reactions producing H₂O₂ can be catalyzed by enzymes bound to the alginate interface, then the generated H₂O₂ diffuses into the alginate hydrogel and partially escapes to the solution.¹⁰⁶ The H₂O₂ fraction that penetrates into the hydrogel is catalytically decomposed to yield free radicals, which disrupt the alginate polymeric chains leading to the release of the loaded species (Fig. 9A). In another configuration, the enzymes or nanozymes can be included in the alginate matrix and produce H₂O₂ internally in the hydrogel resulting in its dissolution and then payload release. The nanozymes^146,147 (catalytic nanoparticles) are large enough for not leaking out prior to the reaction producing H₂O₂. While nanozymes are less specific to a substrate comparing with enzymes, they are more stable providing longer storage and operational time. The enzymes are much smaller and can leak out from the hydrogel prior to its dissolution. In order to keep them inside the hydrogel, the enzymes can be bound to nanoparticles (e.g., SiO₂-NPs; 200 nm diameter) increasing effectively the size of the catalytic species and reducing their leakage (Fig. 9B).

Fig. 9 (A) DNA release from the Fe³⁺–Alg hydrogel triggered by H₂O₂ produced through biocatalytic reactions (Adapted from ref. 106. Copyright 2017 Wiley-VCH). (B) Insulin release from the Fe³⁺–Alg hydrogel triggered by glucose (adapted from ref. 143. Copyright 2017 Wiley-VCH). Abbreviations used in the figure: GOx – glucose oxidase; Glc – glucose; GlcA – gluconic acid (glucose oxidation product).

3.3.3. Release stimulated by light. The photoreduction of Fe³⁺ ions to Fe²⁺ in the presence of α-carboxylic acids operating as sacrificial electron donors has been employed to design light-responsive Fe³⁺–Alg hydrogels.⁸⁵ The photochemical reduction of Fe³⁺ resulted in removal of the cross-linking cations (note that the produced Fe²⁺ cations do not cross-link the alginate chains) and dissolution of the hydrogel accompanied by the release of species entrapped in the hydrogel. The combination of lactate and the Fe³⁺–Alg hydrogel prepared in the form of nano-size species was successfully implemented for photo-triggered release of folic acid.¹¹⁰ Similarly, Fe³⁺–Alg films interpenetrated with cellulose were used as photo-responsive matrices for delivery of small molecules of dexamethasone (a corticosteroid drug) and larger BSA protein molecules.¹⁴⁸ While small drug molecules entrapped in the Fe³⁺–Alg hydrogel were leaking fast, the irradiation of the hydrogel with visible light significantly facilitated their release process. The difference between the leakage and light-induced release was even greater for the larger protein molecules, which demonstrated much smaller leakage prior to the irradiation and the fast release after it.¹⁴⁸ Surprisingly, Fe³⁺–Alg hydrogel beads were able to release small molecules (folic acid and Congo Red dye) upon light irradiation in the absence of the sacrificial α-carboxylic acid electron donor.¹⁴⁹ It should be noted that the photophysical and photochemical mechanisms of the light-triggered decomposition of the Fe³⁺–Alg hydrogel leading to the release processes are not fully understood and require additional studies.

3.4. Biomedicine: from in vitro to in vivo applications

Alginate hydrogels as matrices for 2D and 3D cell cultures have been extensively used owing to their biocompatibility and low cytotoxicity.¹⁵⁰In vitro cell culture studies are essential to make a costless drug development process, increase therapeutic efficacy and understanding drug mechanisms of action. In addition, cell growth in alginate hydrogels are of great relevance for tissue engineering and regenerative medicine applications.⁶¹ Alginate hydrogels have been extensively used for cell culture studies due to their high water content, porosity (diffusion of nutrients in and waste out) and optical transparency important for microscope analysis. Cell culture matrix kits based on alginate hydrogels are commercially available confirming their feasible present and future applications in this field.

Despite the fact that the vast majority of the cell culture studies have been performed on Ca²⁺-cross-linked alginate hydrogels, there is a growing interest in the use of the Fe³⁺–Alg hydrogels as matrices for the 2D and 3D cell cultures in the last few years. In a pioneer study, the effect of the alginate biopolymer composition on the growth of human dermal fibroblasts (HDF) was studied.¹⁵¹ It was found that the Fe³⁺–Alg hydrogels with the high content of G and M subunits have similar efficacy for promoting cell proliferation (Fig. 10A and B). However, the Fe³⁺–Alg hydrogel with the high M content required additional stabilization in cell culture media for 8 days.¹⁵¹ Then, the in vitro cell adhesion was analyzed for the alginate hydrogels cross-linked with Fe³⁺ and Ca²⁺ cations. While the Fe³⁺–Alg hydrogel demonstrated good proliferation of cells, the Ca²⁺-cross-linked alginate hydrogel did not.¹⁵² Vitronectin, a glycoprotein responsible for cell adhesion, migration, and proliferation on different culture media,¹⁵³ plays an important role in cell culturing on alginate hydrogels. Therefore, the adsorption of vitronectin on alginate hydrogels of different compositions was studied. The high hydrophilicity of the Ca²⁺–cross-linked alginate makes it less capable of vitronectin adsorption, while the more hydrophobic Fe³⁺–Alg, demonstrated enhanced vitronectin adsorption.¹⁵² This conclusion was further supported by the measurements of wettability and roughness of both Ca²⁺ and Fe³⁺ cross-linked alginate hydrogels.⁷¹ The obtained results demonstrated that the Fe³⁺–Alg is a promising alternative to the Ca²⁺–cross-linked alginate hydrogel, improving cell adhesion and growth. In addition, harvesting and retrieving of cultured cells can be easily achieved by the dissolution of the alginate matrix using chelating agents such as citrate.¹⁵⁴ Furthermore, the Fe³⁺–Alg was used as a scaffold for 3D growth of cells.¹⁵⁵ Cells were viable inside the matrix up to 14 days after cultivation.

Fig. 10 (A) Proliferation of HDF on Fe³⁺–Alg films under stable gel conditions. (B) Cell number in both High-G and High-M Fe³⁺–Alg films composed of mostly G and M units in the alginate polymer, respectively (adapted from ref. 151 with permission).

In addition to cell growth, the Fe³⁺–Alg hydrogels have been employed for other biomedical applications, typically in combination with other materials. For example, the combination of hyaluronic acid (HA) and the Fe³⁺–Alg hydrogel has found useful applications. On one hand, injectable hydrogels with shear-thinning and antimicrobial activities were developed and the release of Fe³⁺ ions lead to potential long-term treatment of different bacteria, such as Escherichia coli or Staphylococcus aureus.¹⁵⁶ On the other hand, HA–Fe³⁺–Alg hydrogels showed excellent biocompatibility in a mesothelium cell line and efficacy in reducing adhesion formation in a rat model.¹⁵⁷ Alginate–acrylamide hybrid gels cross-linked with ferric ions showed biocompatibility and promising results for tissue engineering.¹⁵⁸ Chondrogenic cells were successfully grown in this matrix. Remarkably, a 2-fold increase in the production of sulfated glycosaminoglycans was found in the hydrogels when irradiated by light. Additionally, polypyrrole-containing Fe³⁺–Alg demonstrated excellent photothermic conversion properties enabling effective tumor hyperthermia treatment in mice.¹⁵⁹

3.5. Environmental remediation

3.5.1. Organic dye degradation. Every year, the production of tons of dyes, mainly for textile, paper and cosmetic industries, generates immense amounts of polluted wastewater.¹⁶⁰ The harmful effects of dyes on health and the environment have attracted interest in developing water treatment technologies.¹⁶¹ Among these techniques, Advanced Oxidation Processes (AOPs) provide an effective and less expensive strategy to degrade dyes under mild pressure and temperature conditions¹⁶² AOPs employ free radicals for an electrophilic attack of organic pollutants resulting in CO₂, H₂O and salts.¹⁶³ The Fenton reaction of iron ions (Fe²⁺/Fe³⁺) and H₂O₂ is an effective approach for hydroxyl free radicals (˙OH) production. Drawbacks in the application of the Fenton reactions are the limited pH range (pH 2–4) and the difficulty and cost of iron recovery from the solution.¹⁶⁴ Different approaches using materials such as fibrous polymers,¹⁶⁵ Nafion membranes¹⁶⁶ or clays¹⁶⁷ have been explored to immobilize iron ions and overcome these limitations. Among other materials, the Fe³⁺–Alg hydrogel beads were successfully used as the immobilization matrix for Fe³⁺ cations as a heterogeneous dye degradation catalyst.¹⁶⁸ Orange II dye was degraded in less than an hour in the presence of H₂O₂ and the Fe³⁺–Alg beads at nearly neutral or slightly acidic pH values (pH 7.8 and pH 5.6). Even faster degradation kinetics was obtained under visible light irradiation¹⁶⁹ (photo-Fenton reaction¹⁷⁰). Notably, control experiments have clearly demonstrated that the presence of H₂O₂ was insufficient for the dye decolorization in the absence of the Fe³⁺–Alg beads, thus, confirming the mechanism of the decolorization process as the photo-Fenton reaction. The photo-Fenton decolorization reaction rate was slower as the number of azo units in the dye increases and the degradation was accelerated by increasing the light irradiation intensity.

In other experiments, Fe³⁺–Alg hydrogel films were employed by Quadrado and coworkers for decolorization of azo Methyl Orange dye.⁶⁴ The films were reused in five consecutive degradation cycles with a slight efficiency decrease. The authors reported an optimal concentration of 1 mM H₂O₂ while higher concentrations of H₂O₂ resulted in loss of degradation efficiency. One could expect that the higher concentration of H₂O₂ the faster would be the dye decolorization. However, at greater concentrations of H₂O₂, the free radical formation (˙OH) can be hindered and H₂O₂ decomposes faster into H₂O and O₂.¹⁷¹ In addition, larger amounts of hydroperoxyl radicals (˙OOH) with lower oxidizing strength might be formed.¹⁷² In a similar study, Fe³⁺–Alg hydrogel fibers were employed as heterogeneous catalyst in the photo-Fenton degradation of Reactive Red 195 dye. The dye degradation was achieved in a wide range of pH 3–9 being faster at acidic pH values. The Fe³⁺–Alg hydrogel fibers were used in four degradation cycles without diminishing decolorization rates.¹⁷³

The use of H₂O₂ is limited by its instability when it is in contact with other chemical species, particularly in wastewater containing multiple residues. The electro-Fenton process¹⁷⁴ is based on continuous electrochemical generation of H₂O₂ to perform the Fenton reaction without the need for H₂O₂ added to the bulk solution. Fig. 11A shows the schematic representation of the electro-Fenton dye degradation using Fe³⁺–Alg beads as the Fe³⁺ ion source. The H₂O₂ is produced via oxygen reduction by bubbling compressed air near the cathode. H₂O₂ reacts with Fe²⁺ electrochemically produced by reduction of Fe³⁺ ions leaked from the Fe³⁺–Alg hydrogel. The Fenton reaction results in free hydroxyl radicals (˙OH) that induce dye degradation. Decolorization of dyes under electro-Fenton process¹⁷⁴ using Fe³⁺–Alg beads has been extensively studied by Sanromán et al.^175–177 Lissamine Green B and Azure B were degraded faster using Fe³⁺–Alg beads as a source of the Fe³⁺/Fe²⁺ ions than by free iron ions added in the system (Fig. 11B).¹⁷⁵ Dyes were degraded in a wide pH range (pH 2–8), degrading faster at acidic pH. The Fe³⁺–Alg beads were reused in 3 cycles, but the time needed for total dye degradation was increased after each cycle, reflecting the decreasing catalytic activity of the Fe³⁺–Alg beads, probably because of decreasing the Fe³⁺ content in the beads. Electro-Fenton oxidation was carried out successfully in a continuous airlift reactor at 3 V and pH 2.¹⁷⁷ However, the system faces a major challenge such as the vigorous agitation of the reactor which promotes Fe³⁺ leakage and hydrogel breakage.

Fig. 11 (A) Schematic representation of an electro-Fenton set up for dye degradation using Fe³⁺–Alg as a catalyst. (B) Degradation of Lissamine Green B and Azure B dyes in the presence of free (in solution) Fe³⁺ and Fe³⁺–Alg hydrogel (adapted from ref. 175. Copyright 2012 Elsevier).

It has been demonstrated that several dye degradation processes can be performed in a wide range of pH values (pH 2–9) using the Fe³⁺–Alg catalyst. This feature has an important environmental implication since most of the residual water from textile industries (pH 7–8) has to be acidified in order to carry out efficiently traditional Fenton reactions, while the acidification is not needed when the Fe³⁺–Alg catalyst is applied. The use of the Fe³⁺–Alg catalyst allows a more economical way for water treatment. In addition to the degradation of dyes, Fe³⁺–Alg hydrogels have been found to be useful for the catalytic degradation of Bisphenol A in aqueous solutions.¹⁷⁸ The Fe³⁺–Alg hydrogels in the form of beads, fibers, and films have noticeable reusability and are easy to handle and remove from solutions. However, leakage of Fe³⁺/Fe²⁺ ions from the catalyst and de-gelation of the Fe³⁺–Alg hydrogel in the presence of H₂O₂ may lead to additional contamination of the wastewater and limits the reusability of the catalyst to a few degradation cycles. Nevertheless, straight-forward procedures have been reported for fast and economical treatment of water polluted by organic contaminants using the Fe³⁺–Alg hydrogels as the catalytic species.

3.5.2. Inorganic contaminant removal. Water contamination by inorganic compounds is a common problem all over the world. The majority of these contaminants (e.g., compounds containing arsenic, chromium, and selenium) are by-products of industry and agriculture.¹⁷⁹ Particularly, arsenic-containing compounds are the greatest mass-poisoning contaminants in the world. The impact of arsenic on health is well known and it is associated with several diseases.¹⁸⁰ Chromium contamination, specially Cr(VI), can cause a wide range of liver and kidney damage, as well as respiratory problems.¹⁸¹ Exposure to selenium over a large period of time is known to have adverse effects, such as the loss of feeling.¹⁸² Techniques for detection, quantification and treatment of these contaminants and others are in the scope of the investigation and numerous applications. Water purification processes based on adsorption/absorption including biochar,¹⁸³ magnetic assistance,¹⁸⁴ nanomaterials,¹⁸⁵ clay minerals,¹⁸⁶ and others^179,187 have been reported and are currently in use.¹⁸⁸

Among these techniques, biomaterials show promising perspectives in cost-effective removal of contaminants from water resources.¹⁸⁹ Calcium-alginate hydrogel has been employed in water treatment for cesium,¹⁹⁰ herbicides,¹⁹¹ copper,^192,193 arsenic¹⁹² and lead¹⁹³ removal. Sorption and removal of different water contaminants using various ionotropic alginate hydrogels, particularly using Fe³⁺–Alg, were studied.^194,195 The comprehensive study included sorption of As(v)–ionic derivatives (arsenates) by alginate hydrogel beads cross-linked with various metal cations: Cu²⁺, Ca²⁺, Fe³⁺, and mixed Ca²⁺/Fe³⁺. The synthesis of the latter was carried out by partial exchange of Ca²⁺ ions by Fe³⁺ ions (Fig. 12A).¹⁹⁶ After 2 hours, Fe³⁺-cross-linked alginate and Ca²⁺/Fe³⁺-cross-linked alginate demonstrated similar adsorption of As(v) ions, but after 24 hours the uptake of As(v) ions was reproducibly larger by the mixed-Ca²⁺/Fe³⁺-cross-linked alginate. Under similar conditions, alginate hydrogels cross-linked with Ca²⁺ and Cu²⁺ ions did not exhibit remarkable As(v) sorption (Fig. 12B),¹⁹⁴ thus clearly demonstrating the advantage of the Fe³⁺-containing alginate hydrogels. In a recent study, arsenate removal efficiencies close to 80% were obtained using Fe³⁺–Alg beads, while Ca²⁺–Alg showed poor removal performance.¹⁹⁷

Fig. 12 (A) Visual appearance of mixed-Ca²⁺/Fe³⁺–Alg beads. (B) As(v) sorption using different ionotropic alginate hydrogels (adapted from ref. 194. Copyright 1998 Elsevier).

Sorption of Se(IV) and Cr(VI) ions by the mixed-Ca²⁺/Fe³⁺-cross-linked alginate hydrogel beads was reported.¹⁹⁵ The removal of Se(IV) ions was comparable with that of As(v) ions, whereas the rate of sorption of Cr(VI) ions was slower and less efficient. The effect of Se(IV), As(v) and Cr(VI) ions competition was analyzed in terms of their sorption by Fe³⁺-Alg.¹⁹⁵ With the same concentration level, removal of As(v) ions and Se(IV) ions remained unaffected by Cr(VI) ions, but on the other hand, the sorption of the latter was reduced by 50% by either As(v) and Se(IV) ions. The competition for sorption between Se(IV) and Cr(VI) ions was obtained due to the similar binding strength of these ions. Similarly, but with different final applications, Fe³⁺–Alg hydrogels have been employed for phosphate¹⁹⁸ and nutrient capture.¹⁹⁹ These results demonstrated the possible use of the Fe³⁺–Alg for agricultural applications.

The literature is consistent with the effects of pH in contaminant removal by ionotropic alginate hydrogels. At very acidic pH <3, the protonation of uronic moieties in alginate decreases the available binding sites. Indeed, the neutral (protonated) sites do not keep Fe³⁺ cations, which are responsible for the sorption of anionic concomitants. In addition, the formation of neutral species of soluble concomitants (e.g., H₃AsO₄, H₂SeO₃) may inhibit their sorption at low pH.^195,196 At basic pH values, the removal efficiency is decreased due to the instability of ionotropic alginate hydrogels. The optimal pH value was found to be pH ca. 4, with a suitable Fe³⁺ ion leakage/contaminant removal ratio.¹⁹⁵ Overall, while the contaminant removal might be efficient enough, particularly with Fe³⁺-containing alginate hydrogels, the exact mechanism of the sorption process requires additional studies.

3.6. Material science

3.6.1. Composite materials. Composite hydrogel materials are produced from two or more individual materials. The major goal of combination of materials is to improve the properties of the original materials, such as porosity, stimuli-responsiveness, stability or mechanical strength. The individual materials can be chemically bound to each other (e.g., upon cross-linking²⁰⁰), electrostatically bound (e.g., produced by the layer-by-layer deposition²⁰¹), or physically interpenetrated between polymeric chains (interpenetrating polymer networks¹¹⁷), or integrated by many other unconventional means.¹¹ Fe³⁺–Alg has been combined with different materials to improve its properties and enhance its performance.

Polyacrylamide interpenetrated in Fe³⁺–Alg has been a widely exploited composite for photoresponsive shape memory material,²⁰² increasing hydrogel stiffness^70,148 stretchability and toughness.²⁰³ Poly(N-isopropylacrylamide) was physically interpenetrated into Fe³⁺–Alg hydrogel conferring it with thermo- and magnetic-responsive capabilities.²⁰⁴ An increase in the iron concentration leads to a significant decrease in pore size and improve in the deswelling rates of the hydrogel. Polypyrrole-containing alginate was used for tumor hyperthermia treatment.¹⁵⁹ The ability of Fe³⁺ ions to synchronously induce gelatinization of alginate and polymerization of pyrrole endowed the material with superior photothermic conversion properties.

The surface porosity of Fe³⁺–Alg hydrogel was significantly reduced after deposition of a polyethyleneimine (PEI) layer cross-linked to carboxylic groups at the surface of Fe³⁺–Alg hydrogel (Fig. 13).¹²⁷ The PEI–alginate composite showed enhanced retention of DNA preventing its premature leakage from the hydrogel. (Fig. 13). Additionally, the incorporation of gold nanoparticles to the hydrogels produced a nanocomposite hydrogel with stimuli-responsive characteristics.¹²⁷ A carbon dot-Fe³⁺–Alg nanocomposite leads to higher absorption in the UV-region showing the potential use of this material for UV-shielding.²⁰⁵ Similarly, cross-linking of sodium alginate with ferric ions containing ethylenediaminetetraacetic acid (EDTA) incorporated within the hydrogel leads to enhanced mechanical properties and flatter and densely packed structure with higher thermal stability.⁸⁷ These properties were necessary for the intended application of the material as UV shielding films.

Fig. 13 Scanning electron (top) and fluorescent confocal microscope (bottom) images of a plain Fe³⁺–Alg and PEI-cross-linked alginate hydrogel (PEI–EDC–Fe³⁺–Alg) (adapted from ref. 127. Copyright 2020 American Chemical Society).

Fe³⁺–Alg was blended with carboxymethyl chitin prior cross-linking with iron ions.⁶⁹ As a result, longer stability and sustained release were achieved in phosphate solutions. A hyaluronic acid (HA) and poly(allylamine hydrochloride) (PAH) layer-by-layer deposition technique was employed to produce photo-responsive PAH/HA/Fe³⁺–Alg nanogels.¹¹⁰ The complexation occurring between PAH and alginate chains hindered the burst release of folic acid that was released in a controlled manner upon light irradiation. Nanofibrous Fe³⁺–Alg strips were synthesized by their combination with polyacrylonitrile (PAN) for tetracycline (TC) visual detection.²⁰⁶ The sensor is based on the complexation of TC with Fe³⁺ which results in a color change easily detected by the naked eye. The sensing strips allowed fast sensing of TC with the possibility to reuse them in 6 detection cycles.

3.6.2. Patterning. Alginate hydrogels biocompatibility and biodegradability make them an ideal biomaterial for regenerative medicine and tissue engineering.⁶¹ For such biomedical applications, the control of the spatial and temporal organization of the polymer without compromising its properties is one of the main challenges.^207,208 Patterning alginate at different length scales is fundamental for in vivo applications as it will allow superior control of the processes occurring in the hydrogel, such as cell growth and leakage of entrapped species. Ca²⁺–Alg patterning strategies are available in the literature.^209,210 The already mentioned mechanical and responsive properties of Fe³⁺–Alg hydrogel have been exploited for patterning of this gel. Photo-patterning is a powerful method for producing hydrogels of specific shapes. Fe³⁺–Alg hydrogel containing lactate was irradiated with light selectively through a mask.⁸⁴ After the dissolution of the irradiated area, the solution was removed obtaining a patterned Fe³⁺–Alg film (Fig. 14A). Then, the patterned Fe³⁺–Alg film was immersed in a 50 mM CaCl₂ and 50 mM ascorbic acid solution, exchanging the Fe³⁺ gelling ions for Ca²⁺ ions (Fig. 14B).²¹¹ Since Ca²⁺-cross-linked alginate cannot be directly photo-patterned because it lacks photochemical properties, the pattering can be done with the Fe ³⁺–Alg, then converting it to the Ca²⁺-cross-linked hydrogel by the cation exchange. Photo-patterning of Fe³⁺–Alg allowed the creation of gradients and interfaces in stiffness and elasticity (Fig. 14C).^158,212 In addition, the photo-dissociable Fe³⁺–Alg coordination was used as a molecular switch to realize photo-control of shape memory on both macroscopic and microscopic scales and enable a number of other functions.²⁰² Fe³⁺–Alg films with complex shapes have been fabricated using templates of patterned paper.²¹³ Chromatography paper was cut with a laser cutter and wetted with the gelling ion solution. The addition of alginate solution resulted in gel formation with the template shape (Fig. 14D).

Fig. 14 (A) Fe³⁺–Alg hydrogel photo-patterned (adapted from ref. 84 with permission); (B) the same hydrogel after exchange of Fe³⁺ for Ca²⁺ cations (adapted from ref. 211. Copyright 2015 Elsevier); (C) Fe³⁺–Alg hydrogel photo-patterned (adapted from ref. 212. Copyright 2015 American Chemical Society). (D) Fe³⁺–Alg and Ca²⁺–Alg growth on templates with different shapes (adapted from ref. 213. Copyright 2009 American Chemical Society).

3.7. Boolean logic operations performed for controlling biomolecule release – Integration of unconventional computing and actuation

Various types of signal-switchable chemical systems have been recently designed for realizing molecule²¹⁴ and biomolecule²¹⁵ computing in the general framework of unconventional computing.²¹⁶ Among many other biochemical systems,¹³⁶ used for biomolecule computing, sophisticated enzyme systems^145,217,218 allowed functional integration of logic operations with actuation functions. These enzyme-based Boolean logic gates allowed control of bioelectronic systems²¹⁹ and molecule-release systems¹⁰⁶ with logically processed biomolecule input signals. Logic control of payload from the Fe³⁺–Alg hydrogel represents one of the systems where the release function is activated with a number of chemical input signals processed by biocatalytic reactions.¹⁴⁴ Silica nanoparticles (SiO₂, ca. 200 nm diameter) were chemically functionalized with different enzymes and then they were entrapped into an Fe³⁺–Alg hydrogel films prepared electrochemically⁴⁷ at an electrode surface. The SiO₂ nanoparticles holding the enzymes and inhibiting their leakage from the hydrogel represent a convenient platform for immobilization of catalytically active enzymes. Fig. 15A shows a reaction cascade catalyzed by lactate dehydrogenase (LDH) and lactate oxidase (LOx) producing H₂O₂ as the reaction output. The input signals, NADH (Input A) and pyruvate (Pyr; Input B) were applied in four different combinations (0, 0; 0, 1; 1, 0; 1, 1) where logic value 0 corresponded to the input absence (physically zero concentration) and logic value 1 was defined as an experimentally optimized concentration of the input chemicals. In order to complete the cascade and produce the H₂O₂ output, both inputs must be present in the system (1, 1 input combination), thus mimicking a Boolean AND logic gate, Fig. 15B. When H₂O₂ was produced, the Fe³⁺–Alg hydrogel film was decomposed in a Fenton-type reaction catalyzed by iron cations yielding free radicals and decomposing the alginate polymer chains,¹⁰⁵Fig. 15C. This resulted in the release of DNA molecules entrapped in the film. The released DNA molecules were analyzed optically (note that they were labeled with a fluorescent dye for a convenient analysis), Fig. 15D. A similar approach was used to perform different logic operations (e.g., Boolean OR logic gate) with the use of different combinations of molecule inputs and different enzymes catalyzing reactions inside the alginate film.¹⁴⁴ The system complexity can be significantly increased by assembling a reaction cascade catalyzed by many enzymes and activated by a larger number of biomolecule input signals. These processes based on multi-step catalytic reactions can mimic Boolean logic operations of high complexity, leading to DNA computers, nano-machines and nano-robots.²²⁰

Fig. 15 (A) The biocatalytic cascade is catalyzed by two enzymes, lactate dehydrogenase (LDH) and lactate oxidase (LOx), yielding H₂O₂ when both input signals, NADH and pyruvate (Pyr), are present. (B) The truth table corresponding to the Boolean AND logic gate. Note that the output signal 1 (corresponding to the H₂O₂ production) appears only in the presence of both input signals (1, 1 input combination). (C) Alginate film decomposition and the DNA molecules release due to the formation of free radicals through a Fenton-type reaction catalyzed by iron cations. (D) Fluorescence analysis of the DNA release (note that it was labeled with a fluorescent dye) upon application of the input signals in different combinations (adapted from ref. 10. Copyright 2020 Elsevier.)

4. Conclusions and perspectives

As it was already noted in the introduction, stimuli-responsive hydrogels are very important functional materials, which can find numerous applications in various areas of science and technology, particularly in biomedical applications.¹⁰ Depending on the properties of specific polymeric materials, the hydrogels can respond to different input signals, physical or chemical, triggering gel–sol transition and releasing a preloaded cargo molecules or nanospecies. On the other hand, the opposite process, the sol–gel transition, can be triggered in the presence of specific cross-linkers or upon changing the environment (e.g., temperature, pH, etc.). While many synthetic polymers have been extensively studied for reversible signal-triggered sol–gel transformations, the natural polymers, and among them alginate, have demonstrated unique mechanical, physical and chemical features, which are especially important for biomedical applications, including signal-triggered drug release. While biocompatibility of alginate is particularly important for biomedical applications,³³ operating as implantable or externally wearable materials, much broader applications in technology have emerged.²²¹ For example, signal-switchable hydrogels (synthetic or natural, including alginate), have been extensively used for creation of signal-switchable interfaces and modified electrodes.²²² Novel unusual applications in biocomputing became possible due to signal-triggered changes in the alginate structure and properties. The important part of the signal-controlled hydrogel structure is a cross-linker that is responsible for the sol–gel transformation. Notably, different metal cations can operate as cross-linkers, each kind of cations with a different functionality. Iron cations (Fe²⁺ ↔ Fe³⁺) possess particularly rich functions changed upon redox transformations, which can be produced chemically, electrochemically, and photochemically. Overall, iron-cross-linked alginate hydrogels have particularly unique properties and ability to respond reversibly to various external signals in biological environments.

The unique features of Fe³⁺–Alg originate from the particular chemistry of Fe³⁺ cations and their exclusive binding to alginate polymer chains. For this reason, an increasing interest in this material has been noted in the number of publications, primarily in the last decade.^223–234 Fe³⁺–Alg can be synthesized from nm to mm scale with different arrangements (e.g., beads, films, electrodeposited layers). In addition, the hydrogel can be degraded by different mechanisms providing the material with stimuli-responsive and patterning capabilities. The stimuli-responsiveness of Fe³–Alg hydrogels has been by far the most appealing feature. It is important to mention that despite the vast amount of responsive materials are available, it is uncommon to find those with multiple-responsiveness. Signals, including H₂O₂, light, pH or electric potential, have been demonstrated to induce hydrogel dissolution and release any encapsulated (bio)molecules and various species (e.g., proteins, nanoparticles, small molecules, etc.) demonstrating “smart” drug delivery processes. The enhanced mechanical properties compared to other ionotropic alginates hydrogels represent an advantage for practical applications in which softer alginate hydrogels are known to have poor performance. Furthermore, the higher hydrophobicity of Fe³⁺–Alg has been exploited for enhanced cell adhesion and growth as well as for contaminant removal.

It should be noted that iron cations, particularly in composition of complex biomolecules, have very important biological functions.^235,236 Therefore, the release of Fe²⁺ cations to biofluids upon dissolution of Fe³⁺–Alg hydrogels may result in physiological changes, which must be considered when biomedical (particularly implantable or invasive) applications are planned.

The use of Fe³⁺–Alg hydrogels, particularly in biomedical applications, has obvious advantages and disadvantages, which in a large extent depend on specific goals and applications. Particularly, the use of Fenton-type reactions producing free radicals for decomposing the alginate matrices and stimulating biomolecule cargo release may result in damaging releasing biomolecules. For example, it has been shown that the DNA released from an alginate hydrogel shows damages because of the catalytically produced oxidative free radicals.¹⁴⁴ To avoid this damage, the loaded DNA was included in a protecting shell. The advantage of the Fe³⁺–Alg hydrogel is its responsiveness to various signals. On the other hand, while it can be dissolved upon application of different signals and their combinations, the hydrogel dissolution and molecule release may proceed non-specifically by a signal which is not properly planned.

Despite the enormous potential of Fe³⁺–Alg hydrogels, their real-life applications are far from now. In our opinion, the following steps have to be done to achieve the practical use of this interesting material: (i) greater efforts need to be done to understand the mechanism by which Fe³⁺ cations coordinate to alginate and other polysaccharides; (ii) mass production of alginate hydrogels is known to be complicated and their heterogeneity have to be addressed to obtain higher reproducibility. This is essential for a better understanding of their behavior in in vivo biomedical applications or environmental applications. For these applications, (iii) it will be required to increase the stability of the hydrogel specifically in environments with high ionic strength, such as biological fluids or residual waters and (iv) enhance the mechanical properties of the hydrogel. The solution to this can be found by the combination of Fe³⁺–Alg with other materials forming composites. As it has been shown before, composite materials using Fe³⁺–Alg are a promising alternative to overcome hydrogels disadvantages and add novel features to the system. In particular, novel advanced materials (metal organic frameworks, graphene, carbon nanotubes or quantum dots) in combination with alginate hydrogels will open several new paths towards smarter, economic and sustainable materials.

Nevertheless, with this review paper, the authors pretend to promote research related with this fascinating material by providing the most updated summary of the properties and highlighting the state-of-the-art of the Fe³⁺–Alg hydrogels.

Data availability statement

The present article is a review of published materials originating from many research groups.

The list of abbreviations (additional abbreviations are explained in figure captions)

AOPs	Advanced oxidative processes
β-Amy	β-Amylase (enzyme)
Ba^2+–Alg	Barium-cross-linked alginate hydrogel
BSA	Bovine serum albumin (protein)
Ca^2+–Alg	Calcium-cross-linked alginate hydrogel
EDC	1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (carbodiimide coupling reagent)
EDTA	Ethylenediaminetetraacetic acid (chelating agent)
Fe^3+–Alg	Fe^3+-cross-linked alginate hydrogel
G	α-L-Guluronate
Glc	Glucose
GlcA	Gluconic acid (product of enzymatic glucose oxidation)
HA	Hyaluronic acid
HDF	Human dermal fibroblasts (main cell type present in skin connective tissue)
Lac	Lactate
LDH	Lactate dehydrogenase (enzyme)
LOx	Lactate oxidase (enzyme)
M	β-D-Mannuronate
MPh	Maltose phosphorylase (enzyme)
NAD^+/NADH	Nicotinamide adenine dinucleotide (oxidized/reduced forms)
NaAlg	Sodium alginate
NPs	Nanoparticles
PAH	Poly(allylamine hydrochloride)
PBS	Phosphate-buffered saline
PEI	Polyethyleneimine
PQQ	Pyrroloquinoline quinone
PQQ-GDH	PQQ-dependent glucose dehydrogenase (enzyme)
Pyr	Pyruvate
TC	Tetracycline

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The authors thank Human Frontiers Science Program for funding through grant RGP0002/2018.

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Footnote

† Deceased in 2021.

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