Hybrid Nanostructured Magnetite Nanoparticles: from Bio-Detection and Theragnostics to Regenerative Medicine

magnetochemistry

Review Hybrid Nanostructured Magnetite Nanoparticles: From Bio-Detection and Theragnostics to Regenerative Medicine

Yolanda Piñeiro *, Manuel González Gómez, Lisandra de Castro Alves, Angela Arnosa Prieto, Pelayo García Acevedo, Román Seco Gudiña, Julieta Puig, Carmen Teijeiro, Susana Yáñez Vilar * and José Rivas Applied Physics Department, Nanomag Laboratory, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] (M.G.G.); [email protected] (L.d.C.A.); [email protected] (A.A.P.); [email protected] (P.G.A.); [email protected] (R.S.G.); [email protected] (J.P.); [email protected] (C.T.); [email protected] (J.R.) * Correspondence: [email protected] (Y.P.); [email protected] (S.Y.V.); Tel.: +34-881813062 (Y.P.); +34-881813062 (S.Y.V.) Received: 5 November 2019; Accepted: 27 December 2019; Published: 10 January 2020

Abstract: Nanotechnology offers the possibility of operating on the same scale length at which biological processes occur, allowing to interfere, manipulate or study cellular events in disease or healthy conditions. The development of hybrid nanostructured materials with a high degree of chemical control and complex engineered surface including biological targeting moieties, allows to specifically bind to a single type of molecule for specific detection, signaling or inactivation processes. Magnetite nanostructures with designed composition and properties are the ones that gather most of the designs as theragnostic agents for their versatility, biocompatibility, facile production and good magnetic performance for remote in vitro and in vivo for biomedical applications. Their superparamagnetic behavior below a critical size of 30 nm has allowed the development of magnetic resonance imaging contrast agents or magnetic hyperthermia nanoprobes approved for clinical uses, establishing an inflection point in the field of magnetite based theragnostic agents.

Keywords: magnetite; superparamagnetism; biodetection; magnetofection; imaging; therapy; tissue engineering

1. Introduction Magnetism has been technologically exploited for centuries, well before quantum mechanics helped to unveil the fundamental mechanisms governing the behavior of magnetic materials. Electrical steels, permanents magnets, nickel-iron alloys or soft ferrites mechanized in different configurations just from bulk, ribbon or disks are the enabling materials behind disparate developments like compasses for marine navigation, bulk magnetic separation devices in mining industry, inductive heating in massive foundry industry,sophisticated devices for electric power generation and distribution or communications and information storage in hard disks [1]. The large variety of applications exploiting different aspects of magnetism has permeated our technically developed society in the last decades and follows still an intense evolution by the hand of applications based on magnetic materials tailored at the nanometric scale. In fact, the optimization and maturity of chemical synthetic procedures during the last decades has allowed the development of materials with designed properties which are only observable at the nanoscale such as, surface plasmonic resonance, enhanced and specific catalytic activity, size-dependent fluorescence,

Magnetochemistry 2020, 6, 4; doi:10.3390/magnetochemistry6010004 www.mdpi.com/journal/magnetochemistry Magnetochemistry 2020, 6, 4 2 of 27 superparamagnetism [2], quantum tunneling magnetization [3] or enhanced coercivity [4] that, all combined, open the door to highly interesting biomedical and technological applications. In addition to their designed properties, magnetic nanoparticles (MNPs) are an intense topic of research in diagnostic, theragnostic and regenerative medicine applications, due to their small size, which is comparable to relevant cell length scales and allows to interact and interfere with biological processes, minimizing adverse effects and opening the way to new diagnostic and therapeutic paradigms [5]. Specifically, superparamagnetic iron oxides nanoparticles (SPIONs) are witnessing a predominant role in nanomedicine developments relaying on their biocompatibility, unbeatable low cost production, physicochemical performance and versatile chemistry that make them almost universally present as a main components in contrast agents for magnetic resonance imaging (MRI), magnetic hyperthermia sources or drug delivery nanoplatforms [6]. It is striking, however, that Nature had incorporated crystalline magnetite nanoparticles (NPs) as a strategy for magnetic guiding in small animals, thousands of years before the recent developments of nanotechnology. Magnetotactic bacteria, bees or pigeons are equipped with magnetic dipolar arrays of small Fe3O4 NPs, biologically synthesized in specific vesicles, that serve as natural compasses to orientate in the magnetic field of the Earth. Moreover, magnetite NPs can be found also in humans, when a pathologically altered iron metabolism triggers their synthesis inside ferritin (Fn), a spherical hollow protein in charge of delivering Fe3+ to feed our cell biomachinery [7]. Besides its good magnetic performance (Curie temperature well above any biomedical application, large values of initial susceptibility and saturation magnetization) and biocompatibility, the availability of facile wet chemistry techniques to produce superparamagnetic (SPM) cores with well controlled size, shape and composition has promoted magnetite to the most relevant position in the field of nanomedicine for biodetection, imaging and therapy. In fact, it competes with the most well accepted soft organic compounds, like liposomes or nano-emulsions, fully biocompatible, biodegradable with enhanced ability for encapsulating a variety of hydrophilic or lipophilic drugs [8] that have found approval for clinical uses in a variety of applications as vaccines, antifunghical, anesthetics or antibiotics [9]. Although with a slower pace, a few set of superparamagnetic magnetite NPs have been clinically tested and approved for commercialization as enhanced contrast agents for magnetic resonance imaging (Feridex; Resovist, Ferumoxtran) [10] or magnetic hyperthermia therapies for brain tumor treatment (NanoTherm) [11], establishing an inflection point in the use of inorganic materials as theragnostic agents. By simple and scalable methods like co-precipitation, hydrothermal or solvothermal decomposition procedures, easy production of spheres, cubes, hexagons, octahedra, hollow spheres, rods, plates or wires can be obtained with controlled size. Combined with surface functionalization procedures, these magnetite NPs can be engineered to produce multifunctional hybrid nanostructures with a designed composition of inorganic/organic/biological shells containing carbon, metal (Au, Ag, Cu, etc.), metal oxides (Ti, Si, Zr, etc.), hydroxides (Al, etc.), organic compounds (polymers like polyacrylic acid (PAA), polyethylene glycol (PEG), etc.), short organic molecules (OAc, dopamine, etc.) and biological moieties (antibodies, aptamers, plasmids, etc.) [12]. This core-shell strategy has given rise to different configurations like single- and multi-core@shell nanoparticles, where magnetite is located in the core (Fe3O4@SiO2; Fe3O4@C), in the shell (gelatin-NPs@Fe3O4-NPs) or embedded in a polymer matrix (polyester, gelatin magnetic beads). In all cases, the nanostructure inherits a combination of abilities that ensures their multimodal capacities for simultaneous magnetic separation/detection/targeting procedures like contrast agents in magnetic resonance imaging/positron emission tomography (MRI/PET), magnetic hyperthermia (MH)/drug delivery therapeutic agents, among others [12]. Moreover, magnetite based nanostructures can be also included as the magnetic phase in a nanocomposite material like mesoporous silica, biopolymer sponges (chitosan, k-carrageenan, alginate, etc.), porous stiff materials (hydroxyapatite, polycaprolactone, etc.) or hydrogels, allowing to produce MagnetochemistryMagnetochemistry2020 2019,,6 5,, 4 x FOR PEER REVIEW 33 ofof 2727

inherited from magnetite, together with magnetic cell growth stimulation or magnetic external magneticfixation [13] sca. ffolds for tissue engineering combining all the theragnostic abilities, inherited from magnetite,The list together of nanostructured with magnetic materials cell growth containing stimulation magnetite or magnetic NPs is externalhuge, and fixation the applications [13]. in nanomedicineThe list of nanostructured cover almost any materials aspect containing from detection, magnetite diagnosis NPs is huge,and therapy and the to applications regenerative in nanomedicinemedicine. cover almost any aspect from detection, diagnosis and therapy to regenerative medicine. InIn thisthis work, work we, we present present the structure, the structure, properties, properties synthetic, syntheti proceduresc procedures and applications and applications emerging fromemerging the magnetically from the magnetically intrinsic properties intrinsic of magnetite properties nanostructures of magnetite with nanostructures tailored configuration, with tailored size andconfiguration, shape. size and shape. 2. Magnetite: Structure and Properties from Bulk to Nanoscale 2. Magnetite: Structure and Properties from Bulk to Nanoscale Magnetite, Fe3O4, is an iron oxide compound where iron ions (with valence 3+ and 2+) adjust Magnetite, Fe33+O4, is2 +an iron3+ oxide compound where iron ions (with valence 3+ and 2+) adjust to to the AB2O4 = Fe (Fe Fe )O4 formulation, arranged in an inverse-spinel crystal structure [14], the AB2O4 = Fe3+ (Fe2+Fe3+)O4 formulation, arranged in an inverse-spinel crystal structure [14], composed by tetrahedral, A, and octahedral, B, sublattices. The magnetic and electric properties of composed by tetrahedral, A, and octahedral, B, sublattices. The magnetic and electric properties of magnetite arise from the interactions between the Fe3+ (d5) and Fe2+ (d6) ions placed on octahedral magnetite arise from the interactions between the Fe3+ (d5) and Fe2+ (d6) ions placed on octahedral positions and Fe3+ (d5) ions on tetrahedral positions. The spinel is a stable crystal structure that accepts positions and Fe3+ (d5) ions on tetrahedral positions. The spinel is a stable crystal structure that accepts the substitution of the A and B lattice locations by a variety of nearly 30 different metal ions with the substitution of the A and B lattice locations by a variety of nearly 30 different metal ions with valences ranging from +1 to +6. An example of this stability is the fact that both, natural magnetite is valences ranging from +1 to +6. An example of this stability is the fact that both, natural magnetite is commonly found containing impurity ions (Ti, Al, Mg, and Mn), and substituted ferrites containing commonly found containing impurity ions (Ti, Al, Mg, and Mn), and substituted ferrites containing transition metals (Co, Mn, Zn) can be easily obtained by wet chemistry procedures [15]. The unit transition metals (Co, Mn, Zn) can be easily obtained by wet3 chemistry procedures [15]. The unit2 cell cell in bulk Fe3O4 consists in a face centered cubic, fcc, (Fd m space group) arrangement of O − ions, in bulk Fe3O4 consists in a face centered cubic, fcc, (Fd3m space group) arrangement of O2− ions, in in which Fe3+ (d5) cations occupy 1/2 of the tetrahedral interstices, and a 50:50 mixture of Fe3+ (d5) which Fe3+ (d5) cations occupy 1/2 of the tetrahedral interstices, and a 50:50 mixture of Fe3+ (d5) and and Fe2+ (d6) cations occupy 1/8 of the octahedral interstices, with a characteristic unit cell parameter Fe2+ (d6) cations occupy 1/8 of the octahedral interstices, with a characteristic unit cell parameter ≈8.4 8.4 Å (see Figure1). ≈Å (see Figure 1).

[111]

Double exchange (FM) [001] B octahedral Fe3+ (d5) Fe2+ (d6)

1250: Superexchange (AFM)

A tetrahedral Fe3+ (d5) [010] a=8.34Å [100]

Figure 1. On the left, the inverse spinel structure (crystal structure adapted from [16] (Science Direct, 2 3+ 5 2016),Figure of 1. FeOn3O th4 eis left, based the oninverse a face spinel centered structure cubic (crystal (fcc) arrangement structure adapted of O − fromions [16] in which (Science Fe Direct(d ), cations2016), of occupy Fe3O4 1is/ 2based of the on tetrahedral a face centered interstices, cubic and(fcc) aarrangement 50:50 mixture of ofO2 Fe− ions3+ (d in5) which and Fe Fe2+3+(d (d65)) cationscations occupyoccupy 11/2/8 ofof thethe octahedraltetrahedral interstices. interstices, Onand the a 50:50 right, mixture the set ofof exchangeFe3+ (d5) and interactions Fe2+ (d6) cations between occupy Fe3+ 5 2+ 6 2 3+ 5 2+ (d1/8), of Fe the (doctahedral) and O −intersticesions, giving. On rise the to right, ferrimagnetic the set of ordering.exchange interactions between Fe (d ), Fe (d6) and O2− ions, giving rise to ferrimagnetic ordering. 2+ 3+ At temperatures above 118 ◦K, Fe and Fe ions are randomly distributed over the octahedral sites allowingAt temperatures electron above hopping 118 between °K, Fe2+ themand Fe and3+ ions giving arerise randomly to the low distributed electrical over resistivity, the octahedral around 3+ 2+ 7sites mili-Ohm allowing/cm electron at Troom hopping, of magnetite between [15 them]. Below and giving 118 ◦K, rise the to Fe the lowand electrical Fe ions resistivity become ordered, around following7 mili-Ohm/cm a cubic at to T orthorhombicroom, of magnetite crystallographic [15]. Below transition, 118 °K, the as Fe proposed3+ and Fe by2+ ions Verwey become [17], where ordered c axisfollowing of the a new cubic orthorhombic to orthorhombic phase crystallographic is parallel to and transition slightly, smalleras proposed than by the Verwey cube edge [17] of, where the fcc c phaseaxis of and the accompaniednew orthorhombic by electronic phase is charge parallel ordering to and of slightly the Fe 3smaller+ and Fe than2+ ions the oncube the edge B sites, of whichthe fcc producesphase and a largeaccompanied increase by in resistivityelectronic [charge18]. ordering of the Fe3+ and Fe2+ ions on the B sites, which producesThe magnetic a large increase ordering in inresistivity magnetite [18] is. dominated by the speciﬁc distribution of Fe3+ and Fe2+ ions onThe both magnetic A and ordering B sites, andin magnetite the exchange is dominated interactions by originatedthe specific when distribution the 3d electronof Fe3+ and orbital Fe2+ ions on both A and B sites, and the exchange interactions originated when the 3d electron orbital of the Fe ions overlap with the 2p electron orbital of O2− ions, since direct exchange between Fe-Fe

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2 of the Fe ions overlap with the 2p electron orbital of O − ions, since direct exchange between Fe-Fe interactions are negligible due to the large distance between Fe ions [19]. Fe-O-Fe super-exchange 3+ 2+ 2 interaction depends on the distance and angle of bonds between the Fe and Fe and O − ions and is responsible for antiparallel alignment of the net magnetic moment of A and B sublattices, which gives rise to the ferrimagnetic order in magnetite [15]. As proposed by Neel, Fe3+ ions on the oppositely aligned tetrahedral and octahedral cancel each other, and only the remaining magnetic moment of Fe2+ ions contributes to the total magnetic moment of magnetite (see Figure1). Moreover, the spin magnetic moments, which are tied to their electron orbit by the spin–orbit interaction, orientate themselves into specific directions in the lattice that minimize the crystalline field and give rise to the magnetic anisotropy axes. In magnetite, easy (low-energy), hard (high-energy) and intermediate (medium-energy) magnetocrystalline anisotropy directions are defined by the cubic lattice axes [111], [100] and [110], respectively (see Figure1)[ 15]. At low temperatures, 118 ◦K, the crystallographic transition from cubic to orthorhombic provokes the appearance of a magnetic isotropic point where the anisotropy constant (K1) sign changes from to +, (K1 = 0) and is known as the − Verwey transition. Compared to other permanent magnetic materials (see Table1), [ 20] magnetite shows a low effective magnetocrystalline anisotropy constant, high saturation magnetization per unit mass (MS~92 emu/g), low coercivity (HC = 10–40 mT) and high Curie temperature (TC = 850◦) compared to relevant temperatures for biomedical applications, and a molecular magnetic moment of 4.1 µBohr (accounting for the larger Fe2+ contribution) [15].

Table 1. Bulk magnetocrystalline anisotropy constant, Kan, Curie temperature, Tc, for representative magnetic materials. Data compiled from data included in [20].

Material Fe Co Ni PtFe Fe3 O4 γ-Fe2O3 3 Kan (kJ/m ) 48 530 4.8 6600 11 4.6 TC (K) 1043 1388 631 750 858 863

However, besides the specific crystalline structure of different magnetic materials, there is one common fact that affects their main magnetic behavior: size. In order to accommodate all the magnetic interaction terms (exchange, Zeeman, demagnetizing field and anisotropy) bulk materials attain stability by adopting a magnetic configuration of multidomains separated by domain walls, that minimizes the total magnetic free energy [21]. When preparing small particles with sizes down to the nanometer scale, the balance of magnetic interaction terms changes: the energy cost of introducing domain walls is higher than the reduction of demagnetizing field, the anisotropy energy decreases proportionally to the reduction in volume of the particle, and single domain becomes the most stable magnetic configuration [22]. This multidomain to single domain transition happens for particles with a critical radius in the nanometer scale, 2 (RC = 10–100 nm), and depends on the material properties as RC = 36 √AK/µ0Ms (A, exchange constant, K, anisotropy constant and Ms, saturation magnetization) [23]. Fe3O4 NPs undergo a transition from multi- to single-domain magnetic structure 80–90 nm, and by further size reduction, which in magnetite happens between 25 to 30 nm, another transition arises when the total anisotropy energy of the crystallite becomes smaller than thermal energy. This fact originates that, in absence of externally applied magnetic fields, the magnetization (M) spontaneously fluctuates at room temperature by thermal stimulation and, in average, M equals zero. This is the so called superparamagnetic regime, characterized by negligible remanence or coercive forces (Figure2a) and reversible magnetization with a rapid “on–off” magnetic switching, modulated by the initial magnetic susceptibility (χin) (in magnetite NPs can range from: χin = 0.5–1) [24]. Magnetochemistry 2020, 6, 4 5 of 27 Magnetochemistry 2019, 5, x FOR PEER REVIEW 5 of 27

75 SQUID 250 50 275

)

4 300

O 3 25 320

-25

M(emu/g

-50

-75 -100 -50 0 50 100 H/T (Oe/K) (a) (b)

FigureFigure 2. 2. (a(a) )Overlapping Overlapping into into a asingle single curve curve of of m magnetizationagnetization data data versus versus H/T, H/T, that that correspond correspond to to measurementsmeasurements performed performed corresponding corresponding from from 250 250 to to 320 320 K, K, on on a a dried dried sample sample of of SPM SPM multi multi-core-core FeFe33OO4@C4@C,, showing showing negligiblenegligible remanence remanence and and coercive coercive forces forces and (b )and Transmission (b) Transmission electron microscopy electron microscopy(TEM) micrograph (TEM) micrograph of multi-core of multi Fe3O-core4 @C, Fe with3O4 @C, averaged with averaged size 187 size nm. 187 (Figures nm. (Figure reprinteds reprinted with withpermission permission from from [25], [2 IEEE,5], IEEE 2016)., 2016).

ChemicalChemical procedures procedures like like coprecipitation, coprecipitation, thermal thermal decomposition decomposition or hydrothermal or hydrothermal preparations, preparations,are the most are used the most wet used chemistry wet chemistry techniques techniques to provide to provide high high quality quality andand monodisperse monodisperse SPIONsSPIONs with with diameters diameters ranging ranging from from 10 to 10 30 tonm 30 that nm have that become have becomea standard a standardmaterial as material magnetic as hyperthermiamagnetic hyperthermia actuators actuators with high with specific high specific absorption absorption rates rates (SAR) (SAR) in in clinical clinical applications applications (MAGFORCE(MAGFORCE-NanoTherm)-NanoTherm) [11] [11 ] or or as as T2 T2 MRI MRI commercial commercial contras contrastt agents agents (Feridex; (Feridex; Resovist, Resovist, FerumoxtranFerumoxtran [10]). [10]). InIn addition, addition, SPM SPM behavior behavior can can be be preserved preserved in in multi multi-core-core configurations configurations (see (see Figure Figure 22b)b) even thoughthough several several magnetic corescores standstand in in close close contact contact embedded embedded inside inside a coatinga coating shell shell (see (see Figure Figure2)[ 252)]. [25].These These NPs NPs are are particularly particularly interesting interesting for for those those applications applications that that require require aa high concentration of of magneticmagnetic material in in a asmall small region region to ensure to ensure an intense an intense magnetic magnetic response response (i.e., cell (i.e., isolation cell purposes). isolation purposes).The key procedure The key consists procedure in coating consists the single-core in coating magnetite the single surfaces-core magn to avoidetite exchange surfaces interaction to avoid exchangebetween theminteraction that may between cause exchangethem that bias, may commonly cause exchange resulting bias, in reducedcommonly saturation resulting magnetization, in reduced saturationlow initial magnetization, magnetic susceptibility low initial or magnetic the loss of susceptibility SPM behavior. or the loss of SPM behavior. However,However, associated associated with with the the size size reduction, reduction, th thee increase increase of of surface surface-to-volume-to-volume ratio ratio (typically, (typically, forfor 5 5 nm nm NPs NPs,, the the surface surface spins spins represent represent 30% 30% of of total total amount amount of of spins spins [2 [266]]),), entails entails the the dominant dominant contributioncontribution of of surface surface properties, properties, incorporat incorporatinging a a crucial crucial problem problem for for magnetic applicationsapplications:: the the reductionreduction of of saturation saturation magnetization magnetization [2 [277]].. For For very very small small magnetic magnetic NPs NPs,, a a surface surface dead dead magnetic magnetic layerlayer appears appears related related to to specific specific features features like: like: 1.1. TheThe large large contribution contribution of of surface surface ions ions,, located located on on edges edges or or corners corners,, with with coordination coordination numbers numbers lowerlower than than inner inner core core ions, ions, (see (see Figure Figure 33)) give givess rise rise to an abrupt breakdown of the lattice and magneticmagnetic symmetry, symmetry, which which induces induces changes changes in in magnetic magnetic anisotropy anisotropy at at the the surface surface.. 2.2. TheThe chemical chemical environment environment of the of thecoating coating shell shellinfluences influences the magnetic the magnetic properties properties of the surface of the ions.surface ions. 3.3. TheThe interaction interaction of of the the surface surface spins spins with with the the inner inner ones ones by by an an exchange exchange bias bias may may lead lead to to some some degreedegree of of frustration frustration that that lowers lowers the the total total magnetization magnetization of of the the nanoparticle nanoparticle [2 [288].].

Magnetochemistry 2019, 5, x FOR PEER REVIEW 6 of 27 Magnetochemistry 2020, 6, 4 6 of 27

S=8 S=26 S=56 1,01.0

0.80,8

VT =8 0,60.6 V =27 S/V T 0.40,4 VT = 64 0,20.2

0,00.0 0 50 100 150 200 250 300 N (ions)

(a) (b)

FigureFigure 3. 3.(a()a Ratio) Ratio of of surface surface to volumevolume ions ions in in a cubica cubic lattice, lattice, for for very very small small nanoparticles nanoparticles (NPs) (NPs surface) surfaceions can ions amount can amount up to up large to large percentages percentages of the of totalitythe totality and and (b) corner,(b) corner, edge, edge, surface surface and and inner inner ions ionswith with diﬀ differenterent coordination coordination numbers. numbers.

AlthoughAlthough the the preserva preservationtion of ofoptimum optimum magnetic magnetic properties properties in insmall small magnetic magnetic particles particles through through thethe increase increase of ofcrystalline crystalline quality quality and and minimization minimization of ofsurface surface distortion distortion has has witnessed witnessed a large a large effort eﬀort inin chemical chemical approaches approaches in inthe the last last years years [29], [29 ],the the production production of ofpure pure colloidal colloidal SPIONs SPIONs remains remains a a challenge.challenge. Mag Magnetitenetite core coress with with a athin thin overoxidized overoxidized (Fe (Fe3+)3 +surface) surface shell shell,, compositionally compositionally close close to toa a maghemitemaghemite polymorph polymorph (γ (-γFe-Fe2O23O),3 are), are normally normally obtained obtained and and only only synthetic synthetic procedures procedures driven driven at athigh high temperaturestemperatures can can improve improve the the purity purity and and crystallinity crystallinity of ofcolloidal colloidal SPIONs SPIONs with with enhanced enhanced saturation saturation magnetizationmagnetization (M (MS = S70=–8570–85 emu/g emu) [30/g)] [.30 ].

3. 3.Synthetic Synthetic Procedures Procedures of ofMultifunctional Multifunctional SPM SPM Magnetite Magnetite Nanostructures Nanostructures ForFor inin vitro vitro oror inin vivo vivo applications,applications, not not only only the the magnetic magnetic core, core, but but also also the the capping capping ligands ligands on on thethe surface, surface, are are the the crucial crucial aspects aspects to toensure ensure an an adequate adequate performance performance of ofthe the magnetic magnetic nanostructures nanostructures whenwhen they they interact interact with with biological biological media, media, like like fluids fluids or or tissues tissues,, where where they they can can be be criticall criticallyy arrested arrested.. However,However, besides besides the theefforts efforts devoted devoted to produce to produce multifunctional multifunctional magnetite magnetite nanostructures nanostructures with highwith quality, high quality, the requirements the requirements to ensure to ensure a good a good in vitroin vitro andand in vivoin vivo performanceperformance include include a a biocompatiblebiocompatible coating coating and and an an adequate adequate solvent solvent dispersant dispersant,, and and for for in invitro vitro andand inin vivo vivo applicationsapplications is iscrucial crucial to to maintain maintain colloidal colloidal stability stability of of the the preparations. preparations. Therefore, Therefore, the the chemical chemical engineering engineering to to prepareprepare SPIONs SPIONs with with several several added added functionalities functionalities requires requires a hierarchical a hierarchical procedure procedure involving involving the the productionproduction of of good good quality quality magnetite magnetite cores andand severalseveral functionalization functionalization steps steps to to add add plasmonic plasmonic NPs, NPs,fluorescent fluorescent moieties, moieties, biological biological agent agent or biocompatible or biocompatible coating coating shells. shells.

3.1. Synthesis of the Magnetic Core. Synthetic Procedures Modulating Size and Shape 3.1. Synthesis of the Magnetic Core. Synthetic Procedures Modulating Size and Shape WithWith the the aim aim of of producing producing magnetite magnetite NPs NPs with with exceptional exceptional magnetic magnetic specifications, specifications, high high crystallinecrystalline quality, quality, well well-controlled-controlled size size and and shape, shape, and and easy easy procedures, procedures, different different wet wet chemistry chemistry approachesapproaches have have been been studied studied based based on on co co-precipitation,-precipitation, thermal thermal decomposition, decomposition, solvo solvothermalthermal or or hydrothermalhydrothermal techniques techniques (summarized(summarized in in Figure Figure4). In4) combination. In combination or alone, or they alone, can be they conveniently can be convenientlymodified to producemodified magnetite to produce NPs magnetite with different NPs morphologies with different (spheres, morphologies cubes, hexagons, (spheres, octahedra, cubes, hexagons,hollow spheres,octahedra, etc.), hollow high spheres, crystalline etc.) quality,, high crystalline different quali architecturesty, different of architectures single- or multiple-core of single- orconfigurations multiple-core configurations [31] or to provide [31] large or to gram-scale provide large production gram-scale of NPsproduction [32]. These of NPs techniques [32]. These have techniquesallowed the have development allowed the of development SPIONs spanning of SPIONs from 10spanning nm to several from 10 hundreds nm to several of nanometers. hundreds of nanometers.

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moderatemoderatecrystallinecrystallinequalityquality goodgoodcrystallinecrystallinequalityquality cheap & scalable low energy procedures high energy procedures cheap & scalable low energy procedures high energy procedures expensiveexpensiveprocedureprocedure shapeshape/size/sizelimitedlimitedcontrolcontrol controlledcontrolledshapeshape

co-precipitation co-precipitation hydro-termal spheres spheresspheres water based hydro-termal spheres [mild[mild(T),(T), P Proom] ] water based room [high[high(T,P)](T,P)] cubes dd<20<20 nm nm cubes octahedraoctahedra thermalthermaldecompositiondecomposition organic [high (T), Proom] hollow spheres organic [high (T), Proom] hollow spheres solventssolvents solvo-termal [high (T,P)] solvo-termal [high (T,P)] multimulti-core@shell-core@shell

FigureFigure 4. 4. Summary Summary of of synthetic synthetic routes routes commonly commonly used used to to produce produce superparamagnetic superparamagnetic iron iron oxides oxides nanoparticlesnanoparticles ( SPIONs (SPIONs).(SPIONs).) .

3.1.1.3.1.1. Precipitation Precipitation Methods Methods PrecipitationPrecipitation methods methods of of iron iron salts salts in in a a highly highly basic basic solution solution are are easy, easy, fast, fast, scalable scalable and and can can be be 2+ 3+ donedone at at low lowlow to toto moderate moderatemoderate temperatures temperaturestemperatures and andand inert inert atmosphere. atmosphere. C Co-precipitationCoo-precipitation-precipitation of of twotwo iron iron FeFe Fe2+2+//Fe/FeFe3+3+ 2+ 3+ saltsalt precursors precursors can cancan be bebe easily easilyeasily controlled controlledcontrolled by byby experimentally experimentally adjusting adjusting the the Fe Fe2+2+//Fe/FeFe3+3+ ions ionsions ratio, ratio, ratio, pH pH and and temperature,temperature, providing providing spherical spherical NPs NPs with with a a good good control control of of the the structural structural (size, (size, morphology) morphology) (see (see FigureFigure 55 5a)a)a) and andand magnetic magneticmagnetic properties propertiesproperties but but a a with withwith wide widewide size sizesize distribution distribution.distribution. .Although AlthoughAlthough its itsits easiness easinesseasiness has hashas made mademade thisthis basic basic method method very very popular, popular, the the ensembles ensembles produced produced in in this this way way inherit inherit a a distribution distribution of of blocking blocking temperatures,temperatures, unde undesiredundesiredsired in in certaincertain certain inin in vivovivo vivoapplications applications applications [33 [3 [3]3 or3] ]or aor distributiona a distribution distribution of magneticof of magnetic magnetic moments moments moments that thatthatneed need need to be to toavoided be be avoided avoided for magneticfor for magnetic magnetic detection detection detection kits. kits. kits.

(a(a) ) (b(b) )

FigureFigure 5. 5. TEMTEMTEM image image image of of of (a( (a)a )) sphericalspherical spherical magnetite magnetite magnetite NPsNPs NPs coated coated coated with with with citrated citrated and and obtained obtained by by a a coprecipitationcoprecipitation method method and and ( b (b) )bare bare cubic cubiccubic magnetite magnetitemagnetite NPs NPsNPs obtained obtainedobtained by byby a aa precipitation precipitationprecipitation method method.method. .

RelatedRelated to to these these needs, needs, optimized optimized optimized co co co-precipitation-precipitation-precipitation procedures procedures have have been been implemented implemented attending attending toto fundamental fundamentalfundamental chemistry chemistrychemistry facts:facts: facts: reactionreaction reaction kinetics,kinetics, kinetics, atmosphere atmosphere atmosphere and and and temperature temperature temperature control. control. control. Following Following Following the thethegeneral general general theory theory theory of nucleation of of nucleation nucleation and growth, and and growth,under growth, the under under so-called the the burst so so-called-called nucleation burst burst approach, nucleation nucleation a monodisperse approach, approach, a a monodispersemonodispersesolution of “nuclei” solution solution can be of of obtained “nuclei” “nuclei” by can can using be be supersaturated obtained obtained by by usingusing solutions supersaturated supersaturated of the precursors solutions solutions to nucleate of of the the in precursorsprecursors to to nucleate nucleate in in a a fast fast and and homogenous homogenous way. way. Further Furtherly,ly, imposing imposing a a slow slow pace pace at at the the growth growth stage,stage, a a monodisperse monodisperse ensemble ensemble of of NPs NPs can can be be achieved achieved [34]. [34].

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Magnetochemistry 2019, 5, x FOR PEER REVIEW 8 of 27 a fast and homogenous way. Furtherly, imposing a slow pace at the growth stage, a monodisperse ensembleIn addition, of NPs since can be magnetite achieved easily [34]. oxides into hematite (Fe2O3), a non-magnetic iron oxide phase, applyingIn addition, inert conditions since magnetite to the reaction, easily oxides with intoa controll hematiteed oxygen (Fe2O3 ),atmosphere, a non-magnetic avoids iron the oxide formation phase, ofapplying Fe2O3 and inert γ- conditionsFe2O3, the other to the ferrimagnetic reaction, with oxide. a controlled oxygen atmosphere, avoids the formation Moreover,of Fe2O3 and crystallineγ-Fe2O3 ,quality the other can ferrimagnetic be optimized oxide. by supplying energy to facilitate the annealing of the magnetiteMoreover, lattice. crystalline A modified quality technique can be [35], optimized based on by the supplying precipitation energy of to a facilitatesingle iron the salt annealing in a basic of solutionthe magnetite and a lattice.free oxygen A modiﬁed atmosphere, technique allows [35 ],to basedobtain on Fe(OH) the precipitation2, which after of heating a single at iron 363 salt K in a waterbasic solution bath for and2 h, aallows free oxygen the oxidation atmosphere, into Fe allows3O4 to to obtain obtain cubic Fe(OH) magnetite2, which NPs after (see heating Figure at 3635b). K in a waterThe bath additi foron 2 of h, allowscompounds the oxidation like dextran, into Fepolyvinyl3O4 to obtain alcohol cubic (PVA) magnetite, PAA, etc., NPs in (see subsequent Figure5b). step of theThe reaction addition ensures of compounds protection like of the dextran, magnetite polyvinyl cores alcohol from oxidation, (PVA), PAA, increase etc., ins biocompatibility subsequent step andof the stabilize reactions the ensures colloidal protection dispersion. of the magnetite cores from oxidation, increases biocompatibility and stabilizes the colloidal dispersion. 3.1.2. Thermal Decomposition 3.1.2. Thermal Decomposition Thermal decomposition of organic iron precursor phase in presence of adequate surfactants (fattyThermal acids, oleic decomposition acid (OA), ofoleylamine, organic iron etc.) precursor, driven phase in high in presencetemperatures of adequate, allows surfactants to improve (fatty the crystallineacids, oleic quality acid (OA), of iron oleylamine, oxide NPs etc.), with driven well incontrolled high temperatures, morphology, allows size to and improve narrow the distribution crystalline quality(see Figure of iron 6) oxide. The NPsreaction with welltemperature controlled is morphology, adjusted to size the and used narrow solvents, distribution which (see are Figure usually6). Thecompounds reaction withtemperature high boiling is adjusted points to (octylamine,the used solvents, phenyl which ether, are phenol usually ether, compounds hexadecan withe highdiol, octadecene,boiling points etc.). (octylamine, phenyl ether, phenol ether, hexadecanediol, octadecene, etc.).

50 nm

(a) (b)

Figure 6. TEMTEM image imagess of ( a) hydrophobic FeFe33O4@OA@OA NPs NPs and and ( b)) hydrophobic cubic Fe33O4@OA@OA NPs, NPs, obtained by thermal decomposition method.

The most commonly used iron organic precursors are iron(III)iron(III) NN-nitrosophenylhydroxylamine-nitrosophenylhydroxylamine (Fe(cup)3),), iron(III) iron(III) acetylacetonate (Fe(acac) 5),), iron iron pentacarbonyl (Fe(CO) 5)),, which which follow follow different different routes: Fe(cup) Fe(cup)33 oror Fe(acac)33 directlydirectly decompose into magnetitemagnetite/maghemite,/maghemite, while Fe(CO)5 goes through an intermediate step of metal formation and then an oxidation of Fe0 intintoo magnetite by addition of a mild oxidant [36].]. ThThisis procedure allows to obtain sophisticated hollow magnetite structures, which in the case of thermal decomposition of iron pentacarbonylpentacarbonyl [[37]37] producesproduces inin aa firstfirst stagestage Fe@FeFe@Fe33OO44 structures ( (seesee Figure7 7a)a) priorprior toto thethe finalfinal formationformation ofof smallsmall hollowhollow magnetitemagnetite spheresspheres (see(see FigureFigure6 6b).b). Large Large hollow hollow magnetite spheres spheres (see (see Figure Figure7c) 7 canc) canbe obtained be obtained through through the thermal the thermal decomposition decomposition of ferric chloride of ferric chloridehexahydrate hexahydrate (FeCl3 6H (FeCl2O) [338·6H]. 2O) [38]. ·

MagnetochemistryMagnetochemistry 20202019,, 65,, 4x FOR PEER REVIEW 99 ofof 2727 Magnetochemistry 2019, 5, x FOR PEER REVIEW 9 of 27

10 nm

10 nm (a) (b) (c) (a) (b) (c) Figure 7. TEM image of (a) Fe@Fe3O4@OA NPs 13 nm, (b) small hollow Fe3O4@OA NPs and (c) STEM 3 4 3 4 FigureFiofgure large 7.7 .hollow TEM image Fe3O4 of@OA (aa)) Fe@FeNPsFe@Fe. 3O4@OA@OA NPs NPs 13 nm, ( b) small hollow FeFe3O4@OA NPs and ( c) S STEMTEM of large hollow Fe33O4@OA@OA NPs NPs.. Thermal decomposition is a versatile procedure that allows also to obtain magnetite NPs with a diminishedThermal magnetic decomposition dead layer is is a a versatile versatile[39], different procedure procedure morphologies that that allows allows like also also spheres to to obtain obtain [40 magnetite] magnetite or cubes NPs NPs[41 ],with withwith a adiminishednarrow diminished size magneticdistribution magnetic dead dead and layer controlled layer [39], [39], different ion diff substitutionerent morphologies morphologies with transition like like spheres spheres meta [ls [4040 like]] or or Fe, cubes Mn, [Co,4141], Ni, with or narrowCr. However, size distribution distribution the use on and andnon controlled- controlledpolar solvents ion ion substitution substitutionis a main disadvantage with with transition transition that meta metalsneedls likes to like Fe,be Fe, overcomeMn, Mn, Co, Co, Ni, by Ni, or a orCrphas. Cr. However,e However, transfer the strateg theuse useony, non onto non-polar- changepolar solvents organic solvents is- stabilizeda ismain a main disadvantage NPs disadvantage into waterthat thatneed dispersions needss to be to overcome besuitable overcome by for a byphasbiomedical ae phase transfer applications. transfer strateg strategy,y, to change to change organic organic-stabilized-stabilized NPs NPs into into water water dispersions dispersions suitable suitable for biomedical applications. 3.1.3. Hydrothermal Synthesis 3.1.3. Hydrothermal Synthesis Hydrothermal synthesis is a case of solvothermal procedures, that submit the reactants into a Hydrothermal synthesis is a case of solvothermal procedures, that submit the reactants into a stainlessHydrothermal-steel autoclave synthesis at high is a pressure case of solvothermal and temperatures procedures but using, that watersubmit as the solvent reactants. This into high a stainless-steel autoclave at high pressure and temperatures but using water as solvent. This high stainlessenergy wet-steel chemistry autoclave technique, at high pressureproduces andNPs temperatures with high crystalline but using quality water and as optimumsolvent. This magnetic high energy wet chemistry technique, produces NPs with high crystalline quality and optimum magnetic energyperformances wet chemistry, since lattice technique, format proionduces benefits NPs f rowithm the high high crystalline temperatures quality (from and optimum355 to 525 magnetic K), high performances, since lattice formation benefits from the high temperatures (from 355 to 525 K), high performancesvapor pressure, sinces (from lattice 0.3 to format 4 MPa)ion and benefits long times from (upthe tohigh 72 h)temperatures to which the ( fromexperimental 355 to 525 conditions K), high vapor pressures (from 0.3 to 4 MPa) and long times (up to 72 h) to which the experimental conditions vaporare subjected pressure. Specifically,s (from 0.3 to a 4combination MPa) and long of high times temperatures (up to 72 h), tobetween which the355 experimentalto 525 K, and conditionsprolonged are subjected. Specifically, a combination of high temperatures, between 355 to 525 K, and prolonged arereaction subjected time. (24Specifically, h), has been a combination studied and of successfully high temperatures applied ,[42] between to produce 355 to Fe5253O K4@OA, and (prolongedsee Figure reaction time (24 h), has been studied and successfully applied [42] to produce3 4 Fe3O4@OA (see reaction8) and Fe time3O4@PAA (24 h), hascoated been NPs studied, with and sizes successfully around 20 applied nm and [42] high to produceyield (nearly Fe O 86%),@OA (bysee mixingFigure Figure8) and3 4 Fe 3O4@PAA coated NPs, with sizes around 20 nm and high yield (nearly 86%), by mixing 8FeCl) and2∙4H Fe2OO and@PAA FeCl coated3∙6H2O NPs in a, Teflonwith sizes vessel around with either 20 nm an and OA highor a PAA yield solution (nearly, respectively.86%), by mixing The FeCl2 4H22O and FeCl3 3 6H2 2O in a Teflon vessel with either an OA or a PAA solution, respectively. FeClso obtained∙4H· O andNPs FeCl show∙6H a· highO in degree a Teflon of crystallinityvessel with either (see Figure an OA 8 )or and a PAA a saturation solution magnetization, respectively. The(MS The so obtained NPs show a high degree of crystallinity (see Figure8) and a saturation magnetizationS so= 84 obtained emu/g, NPsclose show to the a rangehigh degree of bulk of magnetite crystallinity (M (Ssee~92 Figureemu/g )8.) and a saturation magnetization (M (M= 84S =emu/g84 emu, close/g, close to the to range the range of bulk of bulkmagnetite magnetite (MS~ (M92 Semu/g~92 emu). /g).

FigureFigure 8.8. Fe3O4@OA@OA coated coated NPs NPs developed developed by by hydrothermal hydrothermal method method.. ( a) Low Low-resolution-resolution bright-fieldbright-field TEMFigureTEM imagesimages 8. Fe3O andand4@OA correspondingcorresponding coated NPs EDdevelopedED ringring patterns by hydrothermal and (b)) representativerepresentative method. (a HRTEM)HRTEM Low-resolution imageimage togethertogether bright- withfieldwith FourierTEMFourier images Transform Transform and corresponding FT FT pattern pattern (inset). (inset).ED ring White White patterns arrows arrows and ( mark markb) representative the the dimer dimer boundaries. boundaries.HRTEM image Bottom Bottom together insets:insets: with a a filteredFourierfiltered HRTEMHRTEM Transform imageimage FT pattern ofof aa selectedselected (inset). dimerdimer White NPsNP arrowss (leftleft )) mark andand thethe the correspondingcorresponding dimer boundaries. GPA Bottom imagesimages forinsets:for g1g1111̅ 1 a (filtered(middlemiddle )HRTEM) andand g01g011 image1 ̅((rightright of)) didiffractiona ffselectedraction spots.dimerspots. (Figures(NPFigures (lefts reprintedreprinted) and the with withcorresponding permissionpermission GPA fromfrom [images [42]42],, ACS, ACS for, 2014. 2014g111̅ . (middle) and g011 ̅ (right) diffraction spots. (Figures reprinted with permission from [42], ACS, 2014.

MagnetochemistryMagnetochemistry2020 2019,,6 5,, 4x FOR PEER REVIEW 1010 ofof 2727

In another approach, magnetite NPs with rounded cubic shape and 39 nm were obtained by In another approach, magnetite NPs with rounded cubic shape and 39 nm were obtained by coprecipitation at 343 K of ferrous Fe2+ and ferric Fe3+ ions by N(CH3)4OH solution, and a subsequent 2+ 3+ coprecipitationTeflon vessel thermal at 343 K treatment of ferrous at Fe T =and 523 ferric K for Fe 24 hions [43] by. The N(CH improved3)4OH solution, crystalline and and a subsequent magnetic Teflon vessel thermal treatment at T = 523 K for 24 h [43]. The improved crystalline and magnetic quality after the annealing at high temperatures was clearly stated by the increase in MS from 59.8 to quality82.5 emu/g. after the annealing at high temperatures was clearly stated by the increase in MS from 59.8 to 82.5 emu/g. 3.1.4. Solvothermal Procedures 3.1.4. Solvothermal Procedures Solvothermal procedures, using organic solvents at high pressure and temperature, open new Solvothermal procedures, using organic solvents at high pressure and temperature, open new possibilities to prepare magnetite NPs with complex configurations. Sphere like particles with an possibilities to prepare magnetite NPs with complex configurations. Sphere like particles with an average size of 190 nm and containing multiple SPIONs cores closely packed inside a carbon coating average size of 190 nm and containing multiple SPIONs cores closely packed inside a carbon coating shell were prepared using a mixture of ferrocene and acetone and kept at 513 K in a Teflon-lined shell were prepared using a mixture of ferrocene and acetone and kept at 513 K in a Teflon-lined stainless-steel autoclave for 72 h [44]. By means of chemical control, this solvothermal procedure is stainless-steel autoclave for 72 h [44]. By means of chemical control, this solvothermal procedure is suitable to provide NPs with sizes between 100 and 250 nm [45] and morphologically controlled SPM suitable to provide NPs with sizes between 100 and 250 nm [45] and morphologically controlled SPM multi-core Fe3O4@C spheres (see Figure 9). multi-core Fe3O4@C spheres (see Figure9).

200 nm 200 nm

FigureFigure 9.9.SEM SEM micrographs micrographs of SPM of SPM multi-core multi Fe-core3O4 @C Fe3O spheres4@C spher developedes developed by a solvothermal by a solvothermal technique withtechnique diﬀerent with amounts different of amounts hydrogen of peroxidehydrogen ( aperoxide) 0.50, (b ()a 1.00,) 0.50, (c ()b 1.50,) 1.00, and (c) (1.50,d) 2.00 and( mL,d) ensuring2.00 mL , sizeensuring control size (a control) 100, ( b(a)) 150, 100, ((cb)) 200, 150, and(c) 200, (d) and 250 ( nm.d) 250 (Images nm. (Images reprinted reprinted with permissionwith permission from from [45], ACS,[45], ACS, 2011). 2011).

3.1.5.3.1.5. BiogenicBiogenic InspiredInspired ProceduresProcedures MonodisperseMonodisperse magnetitemagnetite NPs,NPs, with preciseprecise shapeshape andand sizesize andand crystallinecrystalline quality,quality, appearappear inin magnetotaticmagnetotatic bacteria,bacteria, honeybees,honeybees, pigeons,pigeons, reptilesreptilesor or amphibians.amphibians. InIn humans,humans, magnetitemagnetite NPsNPs cancan bebe foundfound andand areare the the signature signature of of diseases diseases in in which which an an altered altered homeostasis homeostasis of of iron iron biomineralizes biomineralize NPss NPs at higherat higher rates rates (neurological (neurological disorders, disorders, cancer, cancer, etc.). etc.). The The mechanism mechanism of of magnetite magnetite formation formation inin livingliving organismsorganisms (prokaryotes,(prokaryotes, archaeaarchaea or or eukaryotes)eukaryotes) follows follows three three steps: steps: (1)(1) formationformation ofof aa speciﬁcspecific organicorganic reactorreactor matrixmatrix (e.g., (e.g., vesicles, vesicles, protein protein cages, cages, etc.) etc. with) with favorable favorable chemical chemical environment; environment (2); ( formation2) formation of anof intermediatean intermediate iron iron compound compound and and (3) conversion(3) conversion of this of this iron iron precursor precursor into theinto magnetite the magnetite NPs [NPs46]. [46].

Magnetochemistry 2020, 6, 4 11 of 27

Compared to synthetic routes that occur at high energy operative conditions, natural magnetite biomineralization strategies occur at soft physiological conditions and are inspiring a new paradigm on green chemistry procedures. One of such strategies is based on the use of hollow Fn cages, an ubiquitous protein present in almost all living organisms that contains toxic Fe2+ and transforms it by oxidation into ferryhydrate (FeOOH)—an innocuous iron oxide mineral. In neurological diseases with a biologically altered iron homeostasis, the Fn protein shell transforms its ferryhydrate payload into magnetite NPs by a chemically complex procedure [47]. In a pioneering work [48], empty ferritin (apo-ferritin) was successfully used to synthesize magnetite NPs under high temperature and controlled pH conditions, opening a new bio-mimetic methodology based on the use of hollow biological cavities as templates to perform constrained reactions. Within this approach, cage like structures with sizes in the range from 18 to 500 nm, including virus capsids or ferritins obtained from diﬀerent animal sources, have been used taking proﬁt from their special characteristics (Fn from Pyrococcus furiosus remains stable above water boiling point) in synthetic procedures, at high reaction temperatures, to produce size controlled magnetite NPs [49,50].

3.2. Surface Functionalization: Core Protection, Colloidal Stability, Biocompatibility and Multifunctional Decoration Physiological human media (blood, saliva, etc.) are a crowded biochemical environment with a complex machinery in which foreign materials (viruses, bacteria, etc.) are readily recognized and passivated by the immune system (IS) [51]. Therefore, nanoparticles exposed to in vitro or in vivo conditions require a highly engineered surface to protect the magnetite core from possible oxidation into hematite and ensure their magnetic quality, providing colloidal stability by preventing opsonization (unspecific attachment of a protein corona composed of albumin, immunoglobulin, apolipoproteins) [52] and providing abilities for theragnostic functions. Different capping strategies include the use of small organic molecules, surfactants or active moieties like fluorescent molecules (rhodamine, fluorescein, etc.); natural polysaccharides, such as dextran, artificial block copolymers (poloxamers, poloxamines) or large polymers, like PEG that avoids opsonization [53]; inorganic materials, like amorphous or mesoporous silica and carbon, adding textural properties or thermal insulation; metal NPs (with plasmonic activity) and biological moieties for tagging, penetration or therapeutic abilities (monoclonal antibodies, aptamers, carbohydrates [53] and drugs).

3.2.1. Small Organic Molecules Small organic molecules, or surfactants, can be anchored easily to magnetite NPs surface, since their hydroxyl groups, Fe-OH, greatly facilitate the anchoring of different compounds: alkoxylanes, carboxylic acids, phosphonic acids, dopamine, etc. The most popular in situ coating consists in directly adding small biocompatible compounds (amino acid, citric acid, vitamin, cyclodextrin) during core synthesis. However, additional enhancement of colloidal stability in basic or acidic media, where the organic molecules can decompose, can be achieved by grafting the Fe-OH group surface with silane groups, Si-OCH3, which show good water stability and no cytoxicity [54,55]. Different silane compounds are available, like 3-aminopropyltriethyloxysilane (APTES) and mercaptopropyltriethoxysilane (MPTES), providing chemical versatility [54] since they incorporate amino and sulfhydryl functional groups which facilitate bioconjugation procedures or drug grafting [55,56]. Phosphonic acid forms strong Fe-O-P bonds, which densely graft the magnetite NPs surface and allow a further combination with polydopamine groups, providing an improved pH and temperature stability [56]. Shahoo et al. have reported an efficient coating of 6–8 nm magnetite NPs, by oleic acid, lauric acid, phosphonic acids (dodecyl-; hexadecyl-) and dihexadecyl-phosphate, concluding that the bonding strengths of alkyl phosphonates and phosphates are stronger than that of carboxylate and proposed them as alternative biocompatible coatings in organic solutions [57]. Magnetochemistry 2020, 6, 4 12 of 27 Magnetochemistry 2019, 5, x FOR PEER REVIEW 12 of 27

3 2 7 2 7 2 OleicOleic acidacid (CH(CH3(CH(CH2))7CCHH = CCH(CHH(CH2))7COCO2H)H) provides provides large large colloidal colloidal stability stability to magnetite NPs, butbut itsits lipophiliclipophilic charactercharacter isis aa mainmain drawbackdrawback inin biomedicalbiomedical applicationsapplications (see(see FigureFigure 1010).). ItsIts largelarge 3 2 16 2 stericsteric stability,stability, comparedcompared to to similar similar compounds compounds like like stearic stearic acid acid (CH (CH3(CH(CH2)16) COCO2H), arises fromfrom itsits cis-double-bond,cis-double-bond, whichwhich formsforms aa kinkkink inin thethe middlemiddle ofof thethe carboncarbon chainchain structurestructure [58[58].].

50 nm

Figure 10. TEM images of magnetite NPs coated with oleic acid. Figure 10. TEM images of magnetite NPs coated with oleic acid.

Moreover, reported by Guardia et al. [59], Fe3O4 NPs with sizes from 6 to 17 nm present Moreover, reported by Guardia et al. [59], Fe3O4 NPs with sizes from 6 to 17 nm present large large saturation magnetization values (MS from 79 to 84 emu/g at T = 5 K), close to the bulk value saturation magnetization values (MS from 79 to 84 emu/g at T = 5 K), close to the bulk value (MS = 92 (MS = 92 emu/g), which in contrast to similar bare Fe3O4 NPs of 4 nm have only MS = 50 emu/g. These emu/g), which in contrast to similar bare Fe3O4 NPs of 4 nm have only MS = 50 emu/g. These results results point to the role of oleic acid in reducing the spin surface disorder of small magnetite NPs, point to the role of oleic acid in reducing the spin surface disorder of small magnetite NPs, which is which is of large interest for the magnetic performance improvement in ultra-small SPIONs. of large interest for the magnetic performance improvement in ultra-small SPIONs. To enable lipophilic magnetite NPs [33] for biomedical applications which are mainly water based, To enable lipophilic magnetite NPs [33] for biomedical applications which are mainly water a phase transfer, like the addition of amphiphilic molecules to the oil-soluble phase is the primary based, a phase transfer, like the addition of amphiphilic molecules to the oil-soluble phase is the strategy to create a double layer with the hydrophilic segments exposed towards the solvent [60]. primary strategy to create a double layer with the hydrophilic segments exposed towards the solvent Additionally, surfactant exchange can be afforded by replacing the initial one by a new bifunctional [60]. surfactant, which has one group capable of binding to the NPs surface with a strong chemical bond Additionally, surfactant exchange can be afforded by replacing the initial one by a new and another terminal group that has a polar character and remains exposed to water [60]. bifunctional surfactant, which has one group capable of binding to the NPs surface with a strong Other interesting ability of organic coatings is the protection against degradation in acidic or basic chemical bond and another terminal group that has a polar character and remains exposed to water media. Specifically, it has been reported [61] that magnetite coated with bipyridinium, F O @bipy NPs [60]. 3 4 (13 nm) shows increased water solubility of the ferrofluid up to 300 mg/mL and stability of magnetite Other interesting ability of organic coatings is the protection against degradation in acidic or in a wide range of pH conditions, from very acidic to very basic (1 pH 11), extremely useful for basic media. Specifically, it has been reported [61] that magnetite≤ coated≤ with bipyridinium, acidic in vivo conditions like in tumor locations, where therapeutic procedures require an extended F3O4@bipy NPs (13 nm) shows increased water solubility of the ferrofluid up to 300 mg/mL and period of stability of Fe O NPs. stability of magnetite in3 a4 wide range of pH conditions, from very acidic to very basic (1 ≤ pH ≤ 11), 3.2.2.extremely Large useful Polymers for acidic in vivo conditions like in tumor locations, where therapeutic procedures require an extended period of stability of Fe3O4 NPs. The incorporation of large polymers has been reported to add different advantages like size and shape3.2.2. Large control Polymers in one-pot procedures, enhanced colloidal stability, ability to prevent protein corona formation, biocompatibility and large availability of sites for the bioconjugation of biological moieties (aptamers,The incorporation antibodies). of large polymers has been reported to add different advantages like size and shapeTheir control most in remarkableone-pot procedures fact is that, enhanced they off ercolloidal many repulsivestability, ability groups to balancing prevent protein the attractive corona magneticformation and, biocompatibility Van der Waals and interactions large availability that agglomerate of sites for the magnetite bioconjugation NPs and of numerous biological sitesmoieties for grafting(aptamers, antibodies, antibodies). aptamers, etc. Synthetic functional polymers, such as linear or brush structures like, PEG,Their PVA, most polylactic remarkable acid fact (PLA), is that polyvinilpirrolidone they offer many (PVP) repulsive or PAA, groups (see Figurebalancing 11) arethe commonly attractive incorporatedmagnetic and by Van two der alternative Waals interactions approaches: that grafting agglomerate from and magnetite grafting ontoNPs theand NPs.numerous sites for grafting antibodies, aptamers, etc. Synthetic functional polymers, such as linear or brush structures like, PEG, PVA, polylactic acid (PLA), polyvinilpirrolidone (PVP) or PAA, (see Figure 11) are commonly incorporated by two alternative approaches: grafting from and grafting onto the NPs.

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100 nm 50 nm

(a) (b)

Figure 11. TEM images of magnetite NPs coated with ( a) aminolauric acid and (b) polyacrylic acid (PAA).(PAA).

Grafting polymers on the NPs NPs’’ surface surface,, by by in situ procedures is is the most used strategy, since it allows a strict control of the polymer’s architecture and functionality, and although the density of grating is is poor, poor the, the procedure procedure is easier is easier than than a radical a radical polymerization polymerization departing departing from monomer from monomer growth, whichgrowth ensures, which aensures dense buta dense uncontrolled but uncontrolled coating. coating. In situ PAA graftinggrafting ontoonto FeFe33O4 NPsNPs by by a a one one-pot-pot method method has shown to produce materials with high colloidal colloidal stability stabilit andy andversatile versatile surface surface for grafting for biologicalgrafting moleculesbiological [ 62 molecules]. Other conventionally [62]. Other preparedconventionally one-pot prepared polymer one coated-pot NPs polymer include coated Fe3O NP4@PVPs includ coatede Fe nanocrystals3O4@PVP coated [63], withnanocrystals high colloidal [63], withstability high in colloida 10 differentl stability types in of 10 organic different solvents typesand of organic aqueous solvents solutions, and with aqueous pH ranging solutions between, with pH 2.0 rangingand 11.0. between Generally, 2.0 polyelectrolyte and 11.0. Generally, polymers polyelectrolyte provide enhanced polymers colloidal provide stability enhanced when grafted colloidal onto stabilitymagnetite when NPs, grafted owing on toto their magnetite electrostatic NPs, repulsiveowing to forcestheir electrostatic [64]. repulsive forces [64]. Other than than stability, stability, the the incorporation incorporation of large of polymers large polymers can be used can to be gain used size to and gain morphology size and 2+ 3+ morphologycontrol. In a one-potcontrol. In procedure, a one-pot by procedure, adding PVA by adding to an aqueous PVA to solutionan aqueous of Fesolution/Fe ofsalts Fe2+ with/Fe3+ ureasalts withat 358 urea K, nanosizedat 358 K, nanosized polyhedral polyhedral particles with particles an approximated with an approximated size around size 300 around nm and 300 modified nm and modifiedmicrospheres, microspheres, between 100 between to 280 nm,100 could to 280 be obtained nm, could controlling be obtained the polymer controlling concentration the polymer [65]. Aconcentration much more drastic[65]. A emuchffect onmore shape drastic control effect has on been shape shown control when has two been PAA shown samples when with two average PAA polymerizationsamples with average degrees polymerization of 208 and 126, degree PAA208s of and 208 PAA126, and 126, respectively, PAA208 and were PAA126 used as, respectively an additive, werereactant used to produce as an additive nanorods reactant and flowerlike to produce magnetite nanorods particles, and respectively flowerlike [ 65magnetite]. particles, respectivelBesidesy [65]. enhancing colloidal stability, the specific interaction of the coating polymer with the physiologicalBesides enhancing medium is colloidal of crucial stability, importance the in specific ensuring interaction biocompatibility, of the coating facilitating polymer bioconjugation with the physiologicalor avoiding a fast medium entrapment is of by crucial the IS. Therefore, importance taking in inspiration ensuring biocompatibility, from erythrocytes facilitating (red blood bioconjugationcells) whose chemical or avoiding strategy to a avoid fast entrapment protein corona by formation the IS. Therefore,comprises a taking protective inspiration shell barrier from of erythrocyteshydrophilic oligosaccharide(red blood cells) groups whose [ 66chemical], a neutral strategy electric to avoid surface protein composed corona of formation different hydrophilic comprises acoating protective shells (largeshell barrierbrushes, of densely hydrophilic packed oligosaccharide[67]) have been synthetically groups [66 incorporated], a neutral and electric studied surface [68]. composedIn this context, of different hydrophilic hydrophilic polymer coating brushes shells like linear (large dextrans brushes, and densely their derivatives, packed [67] PEG;) have natural been syntheticallymolecules, like incorporated polysialic acid, and heparin, studied polysaccharides [68]. In this context and artificial, hydrophilic block-copolymers, polymer brushes like poloxamers like linear dextransor poloxamines, and their have derivatives shown to, PEG act as; natural protein molecules corona evaders, like polysialic [69]. acid, heparin, polysaccharides and artificFromial all block these-copolymers possibilities,, like PEG poloxamers coating strategies, or poloxamines known, have as PEGylation, shown to act are as protein the most corona used evadersfor biomedical [69]. applications, and their effectiveness in evading the IS depends on their molecular weightFrom and all density. these possibilities, PEGylated NPs PEG with coating different strategies molecules, known from as 2000 PEGylation and 20,000, are g /themol most and densitiesused for biomedicalbetween 0.5 applications and 50 wt %, haveand their been effectiveness shown to reduce in eva opsonizationding the IS depends from 1600 on counts their molecular per million weight (cpm), andto an density. almost constantPEGylated value NPs of with 400 cpm, different for PEG molecules coating withfrom MW 2000 larger and 20,000 than 5000 g/mo (gl/ mol),and densities showing betweenthat PEGylation 0.5 and can 50 hinderwt % ha butve not been completely shown to stop reduce protein opsonization adsorption from [67]. 1600 Coating counts density per ismillion a key (cpm),factor intocontrolling an almost constant the inter-distance value of 400 between cpm, PEGfor P terminalEG coating brushes with MW that belowlarger athan certain 5000 threshold (g/mol), showinghinder the that adsorption PEGylation of proteins. can hinder Above but not 5% completely of PEG coating, stop protein an inter-distance adsorption threshold [67]. Coating about d 1.0ensity nm isbetween a key factor terminal in PEGcontrolling brushes the can inter be estimated,-distance between and small PEG protein terminal adsorption brushes remains that below constant a certain below threshold400 cpm [ 67hinder]. the adsorption of proteins. Above 5% of PEG coating, an inter-distance threshold about 1.0 nm between terminal PEG brushes can be estimated, and small protein adsorption remains constant below 400 cpm [67].

Magnetochemistry 2020, 6, 4 14 of 27 Magnetochemistry 2019, 5, x FOR PEER REVIEW 14 of 27

Functional polymers with responsive performance under pH, temperature or light irradiation stimuli are essential to provide controlled drug release [70]. Specifically, Specifically, thermoresponsive polymer hydrogels like Pluronic,Pluronic, poly-isopropylacrilamidepoly-isopropylacrilamide ( (PNIPAM)PNIPAM) and their derivatives (e.g. (e.g.,, PNIPAM with chitosan [71])]) can bebe usedused toto expelexpel loadedloaded moleculesmolecules exploitingexploiting theirtheir coil-to-globulecoil-to-globule transition at defineddefined temperatures.temperatures. These These hydrogels, hydrogels, in in combination combination with with SPION SPIONss can can be be heated up by the application of of an an alternating alternating magnetic magnetic field field (magnetic (magnetic hyperthermia) hyperthermia) to undergo to undergo a controlled a controlled network networkshrinking shrinking useful for useful enhanced for enhanced drug delivery drug applicationsdelivery applications [72]. [72].

3.2.3. Inorganic Materials Inorganic materials materials are are used used also also to confer to confer core protection; core protection colloidal; colloidal stability and stability other and functional other propertiesfunctional properties like surface like plasmon surface resonance plasmon (metalresonance NPs), (metal thermal NPs) isolation, thermal (carbon isolation shell), (carbon etc. shell) Silica, (SiO ) is one of the most commonly used inorganic coating materials, due to its versatile chemical etc. Silica2 (SiO2) is one of the most commonly used inorganic coating materials, due to its versatile chemicalsilanol surface silanol groups surface (-SiOH), groups colloidal (-SiOH), stabilitycolloidal and stability biocompatibility. and biocompatibility The preparation. The preparation of magnetite of magnetiteNPs coated NPs with coated silica bywith sol-gel silica procedures by sol-gel procedures allows to control allows the toshell control thickness the shell [73 thickness] and is aff [73]ordable and isby affordable the basic hydrolysis by the basic of silanes hydrolysis in aqueous of silanes solutions in of aqueous different solutions organosilane of different compounds organosilane (tetraethyl compounorthosilicateds (TEOS),(tetraethyl APTES). orthosilicate The reaction (TEOS) between, APTES the). oxide The reaction surface of between magnetite the and oxide the silica surface takes of placesmagnetite by the and OH the− silicagroups takes [74 ],places and by by an the adjustment OH− groups of the[74], amount and by ofan added adjustment TEOS of and the the amount reaction of addedtime, the TEOS silica and shell the thicknessreaction time, can bethe easily silica tailored.shell thickness Moreover, can be di effasilyerent tailored. configurations Moreover, of thedifferent silica configurationscoated magnetite of the nanostructures silica coated canmagnetite be achieved nanostructures following can two be di achievedfferent routes: following Stöber two processes different routes:generally Stöber result processes into a multi-core generally coatedresult into NPs, a multi while-core microemulsion coated NPs processes, while microemulsion provide mainly processes single providecore-shell mainly NPs [ 74single] (see core Figure-shell 12 NPs). [74] (see Figure 12).

100 nm

(a) (b)

Figure 12. TEM micrographs of silica coated magnetite NP NPss by ( a) Stöber proces processes,ses, which generally result in in multi multi-core-core coated coated NP NPss and and (b) ( bmicroemulsion) microemulsion process processes,es, which which provi providede mainly mainly single single core- shellcore-shell NPs. NPs.

4. Biomedical Applications Arising from the Tunable MagneticMagnetic PropertiesProperties ofof MagnetiteMagnetite Nanoparticles Nanoparticles The SPM character, inherent from the magnetite cores, allows to combine several abilities within a The SPM character, inherent from the magnetite cores, allows to combine several abilities within single nanostructure that can be triggered externally, under demand, by the application of a remote a single nanostructure that can be triggered externally, under demand, by the application of a remote magnetic ﬁeld to produce diﬀerent magnetic responses of biomedical interest (summarized Table2) magnetic field to produce different magnetic responses of biomedical interest (summarized Table 2) like magnetophoresis, hyperthermia, magnetization or magnetic resonance. like magnetophoresis, hyperthermia, magnetization or magnetic resonance.

Table 2. Summary of biomedical applications based on the combination of SPIONS and magnetic stimulation.

Magnetic Application Pros/Cons. Commercial Kit Toxicity Property - Selective separation of tagged cells without damaging MACS® - Unaltered sample fluid after No in vitro Magnetophoresis Cell isolation Separators, separation toxicity Miltenyi - Requires the use of antibodies for selective binding

Magnetochemistry 2020, 6, 4 15 of 27

Table 2. Summary of biomedical applications based on the combination of SPIONS and magnetic stimulation.

Magnetic Application Pros/Cons. Commercial Kit Toxicity Property - Selective separation of tagged cells without damaging MACS® No in vitro Cell isolation - Unaltered sample ﬂuid after separation Separators, toxicity - Requires the use of antibodies for Miltenyi selective binding Magnetophoresis - Fast/eﬃcient transfection of magnetized agents No in vitro Magnetofection NONE - Hallbach arrays with permanent magnets toxicity are cheap and easy to implement - Magnetized stents can attract injected no in vivo Magnetic guiding magnetic NPs to reduce restenosis NONE toxicity - In preclinical stage tested in rat - Most sensitive magnetometry for Magnetometry No in vitro iron detection NONE SQUID toxicity - Expensive devices - Detecting surface requires the binding GMR No in vitro Magnetic detection of antibodies NONE sensors toxicity - Versatile for microarray detection - Immunodetection requires the binding Impedance of antibodies No in vitro NONE Sensors - Implemented in paper strips kit toxicity - Attains detection limits in the clinical range

- Selective killing of cancer cells MAGFORCE Hyperthermia - In clinical use for glioblastoma tumors NanoTherm, NONE cancer treatment - Increase of life expectancy 6–13 months (clinical therapy) - Controlled release by magnetic Thermally hyperthermia stimulation Inductive Enhanced release No in vitro - Selective targeting NONE heating of therapeutic toxicity - Under demand dosage agents - Under study stage

- Allows deep brain stimulation avoiding the no in vivo Thermal toxicity implantation of electrodes in brain NONE stimulation tested in - In preclinical stage mouse Ferucarbotran ® MRI - MRI contrast enhancement for soft tissues (Resovist , Bayer contrast agents: Healthcare) NONE - In clinical use single (T2) (MRI clinical Magneticre diagnosis) laxation no in vivo MRI - Enhanced contrast for soft and hard tissues. toxicity contrast agents: - Avoiding the use of Gd for T1 contrast NONE tested in dual(T1/T2) - In preclinical stage mouse - Bioprinting of tissue or replacements with biologically functional - No need of artiﬁcial substrate No in vitro Magnetic 3D magnetic - Bioprinted functional saliva NONE and ex vivo prototyping bioprinting secretory organoids toxicity - Tested ex vivo - Under study stage

4.1. Magnetophoresis Magnetophoresis refers to the controlled motion of SPIONs in a viscous medium induced by the application of an external magnetic field. This useful characteristic can be used to isolate, concentrate and drive magnetically labelled biomarkers or cells, from physiological samples (cerebrospinal fluid (CSF), blood, saliva, cell aspirates, etc.) and for in vivo targeting with the application of an external magnetic gradient. The use of magnetic separation techniques saves energy when exposing analytes to a permanent magnet and includes a high degree of specificity, if magnetic particles are conveniently Magnetochemistry 2020, 6, 4 16 of 27 coated with antibodies that specifically target the desired biomarker. Moreover, extraction of the target can be done, without damaging the rest of the fluid for further analysis.

4.1.1. Cell Isolation Purification and preconcentration of biomarkers or cells from patient’s fluid samples or aspirates are crucial for a successful culture of isolated cells for transplantation or for an accurate high signal-to-noise ratio detection step (magnetic, optical or chemical) without the unspecific interference of other biochemical compounds in the sample [75]. Simple magnetic separation has been described by using cationic liposomes loaded with SPIONs (MCL), which were mixed with bone marrow aspirates under shaking for 1 h and transferred into tissue culture dishes, with a disk-shaped magnet at the bottom. Magnetically labeled cells were subject to the attraction of the magnetic field and 30% of them were isolated. Increased separation effectivity, up to an 85% was afterwards achieved using magneto-liposomes, conjugated with CD105 antibodies [76].

4.1.2. Magnetofection Magnetofection, consists in the transfection of magnetic therapeutic vectors (plasmids, engineered viruses, etc.) inside cells, forced by a magnetic field, and is becoming a widely used technique in gene therapy which benefits from its higher degree of penetration compared to non-magnetic approaches. Polyethylenimine-modified SPIONS/pDNA complexes (PEI-SPIONs/pDNA complexes) have been used to rapidly and uniformly distribute on the surface of MG-63 osteoblasts cells, by their incubation under the exposure to a uniform magnetic field enabled by specially designed Hallbach array of permanent magnets. With this homogeneous magnetic exposure, local transfection, without the disruption of the cells’ membrane, was obtained, improving the magnetofection efficiency of pDNA into osteoblasts, thereby providing a novel approach for the targeted delivery of therapeutic genes to osteosarcoma tissues as well as a reference for the treatment of other tumors [77].

4.1.3. Magnetic Guiding Magnetic guiding of MNPs exposed to the application of a magnetic gradient can be used to deliver therapeutic payloads in a precise location with a high degree of specificity. An innovative approach has been studied to reduce the re-stenosis of vasculature after stenting in in vivo experiments by combining stainless steel stents and the use of paclitaxel loaded SPIONs. The stents were magnetized upon the external application of a magnetic field, creating a magnetic field gradient capable to attract the MNPs delivered to the rat via a catheter. Paclitaxel release to the surrounding tissues was effective, reducing therefore restenosis of the vasculature tissue although the dose of paclitaxel-SPIONs was low [78].

4.2. Magnetic Detection Magnetic detection exploits diﬀerent aspects of the magnetic properties of both SPIONs and the components of the detecting sensors.

4.2.1. Superconducting Quantum Interference Device (SQUID) is the most sensitive magnetometer for DC or low frequency measurements. Constituted by superconducting loops containing Josephson junctions, when a current is induced in the SQUID ring by an external magnetic flux, a change in voltage happens across the junction, which generates an output signal from the amplified voltage [79]. Although SQUID has been used to detect ferromagnetic contamination in the lungs or magnetite NPs brain in AD patients in ex vivo conditions [80], it has been applied to susceptibility measurements for in vivo identification of SPIONs loaded on a tumor in the lymph nodes of rats [81]. Moreover, the effectivity of iron quantification by SQUID magnetic relaxometry was successfully used to evaluate SPIONs conjugated to Her2-expressingMCF/Her2-18 cells (breast cancer cells) injected into xenograft MCF7/Her2-18 tumors in nude mice [82]. Brown Magnetochemistry 2020, 6, 4 17 of 27 relaxation of magnetic NPs in liquids has also been exploited to detect biomarkers in liquid-phase immunoassays using SQUID magnetometry of magnetite beads conjugated with biotin, with sizes from 54 to 322 nm, attaining a sensitivity of 5.6 10 8 mol/mL [83]. × − 4.2.2. Giant Magnetoresistance (GMR) Giant Magnetoresistance (GMR) sensors are based on the significant change in electrical resistance of an arrangement of thin-film layers of alternating ferromagnetic and nonmagnetic conducting spacers when they are exposed to an externally applied magnetic field [84]. The variation in magnetoresistance of these spin-dependent sensors decorated with specific antibodies, provides quantitative analysis in combination with MNPs. The main strategy behind several magnetoresistive detectors, consists in attaching MNPs to the analytes, which during the detection procedure remain attached to the sensing surface creating a magnetic disturbance that triggers the change in electrical resistance of the device [85]. GMR detectors have been improved in the last years, being able to respond even upon small variations of magnetic fields, incrementing their sensitivity to detect small concentrations of analytes [84]. Although accurate GMR detection is facilitated by using SPM magnetite beads [86] with a large magnetic moment, a successful strategy using small SPIONs (30 nm) labeled with streptavidin was developed to detect Interleukin-6 (IL-6) antibody and amine modified DNA (deoxyribonucleic acid) oligonucleotide, by functionalizing the surface of the sensor with APTES and Glutathione [87]. This APTES-Glu modification was successfully extended to microarray detection, showing to be a versatile and robust method [87].

4.2.3. Impedance Based Sensor Emergent strategies are trying to couple lateral flow immunoassay (LFIA) (paper-strips), which stand out for their low-cost, speed, portability and ease of use, with an innovative magnetic detection based on Cu-impedance change in the presence of MNPs [88]. The sensing property relies on the high rate of oscillation of the magnetic moments of the SPIONs that induces localized eddy currents on the surface of the sensor, increasing its electrical radio-frequency impedance, for which no external magnetic field is required. This facile magnetic detection strategy is based on the use of paper-strip immunodetection kits in combination with SPIONS functionalized to specifically bind to prostate-specific antigen PSA. The strips were seeded with capture anti-PSA antibody and anti-IgG (both at a concentration of 1 mg/mL) forming the test and control lines, respectively. PSA standard solutions at known concentrations were submitted to the strip and attached to test line. The subsequent binding of conjugated SPIONs to PSA ensured the magnetic reading and the final determination of analyte concentrations. This facile method allowed to attain a limit of detection within the clinical range of interest around 0.25 ng/mL and a resolution of 50 pg [89]. Further improvements are reported, by using large SPM multi-core carbon coated nanoflowers (Fe3O4@C) as LFIA labels that provide a magnetic moment substantially larger than that of single-core SPIONs, allowing for a more sensitive and precise detection [90].

4.3. Inductive Heating Inductive heating, also known as magnetic hyperthermia, consists in the transformation of electromagnetic energy into heat, when an alternating external magnetic ﬁeld (f = 3 kHz, 300 MHz) is applied to a magnetic nanoparticle. The temperature rise that is transmitted to the medium, in which the NPs are housed, depends on the magnetic quality of the NPs, the viscosity of the medium and the parameters of the external magnetic ﬁeld, among others [91]. This externally controlled rise of temperature can be used for killing cancer cells when rising to 315 K, stimulating cell growth for mild thermal increase or triggering release mechanisms for enhanced delivery. MagnetochemistryMagnetochemistry2020 2019, ,6 5,, 4 x FOR PEER REVIEW 1818of of 2727

4.3.1. Cancer Hyperthermia Treatment 4.3.1. Cancer Hyperthermia Treatment Through magnetic hyperthermia, cancer treatments can be developed by selectively heating Through magnetic hyperthermia, cancer treatments can be developed by selectively heating between 314 and 316 K cancerous tissues which are previously loaded with MNPs. In biomedical between 314 and 316 K cancerous tissues which are previously loaded with MNPs. In biomedical 8 −1 applications, magnetic exciters are restricted to a safety upper limit of H∙f < 4.58 ×∙108 A∙m∙s 1 [92]. applications, magnetic exciters are restricted to a safety upper limit of H f < 4.58 10 A m s− [92]. This technique benefits from a natural mechanism called enhanced· permeation×· an· d· retention, This technique benefits from a natural mechanism called enhanced permeation and retention, due due to the defects and poor drainage of cancerous tissues that cause the accumulation of NPs inside to the defects and poor drainage of cancerous tissues that cause the accumulation of NPs inside the the damaged tissue [93]. This entrapment of NPs inside the tumor tissue allows the heating and damaged tissue [93]. This entrapment of NPs inside the tumor tissue allows the heating and weakening weakening of cancer cells, minimizing side effects in the surrounding healthy cells. It is worth of cancer cells, minimizing side effects in the surrounding healthy cells. It is worth highlighting the highlighting the development by MagForce [94] of a clinically used magnetic hyperthermia therapy, development by MagForce [94] of a clinically used magnetic hyperthermia therapy, which combines a which combines a magnetic field applicator (100 kHz) and an injectable therapeutic agent consisting magnetic field applicator (100 kHz) and an injectable therapeutic agent consisting in Fe3O4@amylosan in Fe3O4@amylosan NPs to treat glioblastoma, an aggressive brain tumor. NPs to treat glioblastoma, an aggressive brain tumor. Within this commercial solution, even large tumors around 5 cm can be treated by the direct Within this commercial solution, even large tumors around 5 cm can be treated by the direct injection with 3 mL of magnetite based ferrofluid, which heats up by the application of the external injection with 3 mL of magnetite based ferrofluid, which heats up by the application of the external alternating magnetic fields. This technique has been used in phase II clinical trials in combination alternating magnetic fields. This technique has been used in phase II clinical trials in combination with stereotactic radiotherapy and obtained the European approval as a treatment for brain tumors with stereotactic radiotherapy and obtained the European approval as a treatment for brain tumors in in 2010, after demonstrating its ability to increase life expectancy from 6 to 13 months in patients with 2010, after demonstrating its ability to increase life expectancy from 6 to 13 months in patients with glioblastoma multiforme, compared to others treated only with chemotherapy. glioblastoma multiforme, compared to others treated only with chemotherapy.

4.3.2.4.3.2. ControlledControlled ReleaseRelease AnotherAnother therapeutic therapeutic strategy strategy based based on magnetic on magnetic hyperthermia hyperthermia consists inconsists the thermal in the stimulation thermal ofstimulation thermo-active of thermo moieties-active to provoke moieties the to release provoke of therapeutic the release agents of therapeutic (drugs, growth agents factor, (drugs, oligomers, growth etc.)factor, under oligomers, demand. etc.) under demand. OneOne of of the the most most successful successful drug drug delivery delivery applications, applications, triggered triggered by by magnetic magnetic hyperthermia, hyperthermia, combinescombines SPIONs SPIONs with with a coatinga coating of thermosensitiveof thermosensitive polymers polymers such such as PNIPAM as PNIPAM and its and derivates. its derivates These. polymersThese polymers form hydrogels form hydrogels at room temperatureat room temperature and can allocate and can molecules allocate acting molecules as drug acting nanocarriers. as drug Whennanocarriers the temperature. When the rises temperature above a critical rises range above (T a= critical306–313 range K), these (T = structures 306–313 retractK), these and structures expel all theretract drug and contained expel all within. the drug contained within. StepsSteps areare beingbeing takentaken soso thatthat thisthis techniquetechnique meetsmeets thethe essentialessential conditionsconditions ofof anan eeffectiveﬀective tooltool inin precisionprecision medicine,medicine, temperature temperature control control and and release release time time control. control. InIn fact,fact, itit hashas alreadyalready beenbeen possiblepossible toto produceproduce thethe collapsecollapse ofof 49%49% ofof thethe volumevolume ofof thethe therapeutictherapeutic agentagent atat aa temperaturetemperature ofof biomedicalbiomedical interestinterest TT= = 311311 KK (see(see FigureFigure 1313)[) [9595].]. OptimizingOptimizing thethe biopolymersbiopolymers mixturemixture (PNIPAM(PNIPAM//chitosan),chitosan), itit isis eveneven possiblepossible toto releaserelease 70%70% ofof the the drug drug housed housed in in a a single single second second [ 71[71].].

1.15 1.08 b

) 1.10 a )

1.03 a.u

1.05 a.u

1.00 0.98 0.95

0.90

0.93 ABSORBANCE ( ABSORBANCE 0.85 ( ABSORBANCE

0.80 0.88 CTR VEGF PNIPAM PNIPAM PNIPAM/38 PNIPAM/38 CTR VEGF PNIPAM PNIPAM PNIPAM/38 PNIPAM/38 +VEGF +VEGF +VEGF +VEGF FigureFigure13. 13. AbsorbanceAbsorbance measurementsmeasurements performedperformed toto addressaddress VEGFVEGF releaserelease forfor HUVECHUVEC cellscells incubatedincubated withwith PNIPAMPNIPAM (release (release temperaturetemperature atat 305305 K)K) andand PNIPAMPNIPAM/38/38 (release (release temperaturetemperature atat 311311 K)K) loadedloaded with VEGF and raised to diﬀerent temperatures, (a) 311 K and (b) 293 K. The wells with standard with VEGF and raised to different temperatures, (a) 311 K and (b) 293 K. The wells with standard cultural medium with and without VEGF (10 ng/mL) were used as controls. Experiments performed cultural medium with and without VEGF (10 ng/mL) were used as controls. Experiments performed at 311 K show the ability of thermal release of VEGF loaded on PNIPAM based materials. (Images at 311 K show the ability of thermal release of VEGF loaded on PNIPAM based materials. (Images reprinted with permission from [95], Springer, 2014). reprinted with permission from [95], Springer, 2014).

MagnetochemistryMagnetochemistry 20192020, ,56, ,x 4 FOR PEER REVIEW 1919 of of 27 27

Besides drugs, there is a large interest in the controlled release of biological moieties with therapeuticBesides effect drugs,s for there regenerative is a large interest medicine in. the Several controlled proteins release (recombinant of biological human moieties bone with morphogenictherapeutic e ff proteins,ects for regenerative rhBMPs, induce medicine. osteogenesis) Several proteins and (recombinant growth factors human (vascular bone morphogenic endothelial growthproteins, factor rhBMPs, (VEGF) induce) responsible osteogenesis) for and the growth triggering factors of (vascular angiogenesis endothelial (process growth of factorblood (VEGF)) vessel formationresponsible) are for the crucial triggering for ensuring of angiogenesis the long (process-term of viability blood vessel of repaired formation) tissue ares crucial with for functional ensuring recoverythe long-term. Specifically, viability VEGF of repaired is effective tissues only with when functional concentration recovery. Specifically,gradients at VEGFphysiological is effective levels only arewhen generated concentration, and new gradients strategies at physiological are needed levelsfor an are adequate generated, delivery and new at the strategies site of aretissue needed repair for. In an thisadequate context delivery, the atgraftin the siteg ofcomplex tissue repair.-elastin In- thisdendron context,-VE theGF graftinghyperbranched complex-elastin-dendron-VEGF poly(epsilon-lysine) peptideshyperbranched integrating poly(epsilon-lysine) in their core parallel peptides thermoresponsive integrating inelastin their-like core peptide parallel sequences thermoresponsive over the surfaceelastin-like of Fe peptide3O4@PAA sequences NPs (see over Figure thesurface 14) has of been Fe3O reported4@PAA NPs[96]. (see Figure 14) has been reported [96].

200 nm

(a) (b) (c)

FigureFigure 14. 14. (a()a SEM) SEM micrograph micrograph of of Fe3O4@PAA Fe3O4@PAA-C-ELP-G3K-Bet16-VEGF,-C-ELP-G3K-Bet16-VEGF, (b (b) )h hyperthermiayperthermia Treatment Treatment

ofof Fe Fe3O3O4@PAA4@PAA and and Fe Fe3O3O4@PAA4@PAA-C-ELP-G3K-Bet16-C-ELP-G3K-Bet16 samples samples immersed immersed in in phosphate phosphate buffered buﬀered saline saline,, pHpH 7.4 7.4,, w withith a avolume volume of of 240 240 µµLL and and a amagnetite magnetite concentration concentration of of 40 40 µµg/gµ/µLL,, and and (c (c) )release release of of hVEGF hVEGF

fromfrom Fe Fe3O3O4@PAA4@PAA-C-ELP-G3K-Bet16-VEGF-C-ELP-G3K-Bet16-VEGF after after hyperthermia hyperthermia treatment. treatment. Negative Negative control control shows shows no no hhVEGFVEGF release release after after hyperthermia hyperthermia treatment. treatment. (Images (Images taken taken form form [9 [966],], Elsevier, Elsevier, 2015 2015).).

TheseThese functionalized functionalized SPIONs werewere ableable toto avidly avidly bind bind VEGF VEGF forming forming a stablea stable complex. complex. Under Under the theadequate adequate stimulation stimulation of the of complex the complex Fe3O4 @PAA-C-ELP-G3K-Bet16-VEGFFe3O4@PAA-C-ELP-G3K-Bet16- withVEGF a magneticwith a magnetic induction induction(H = 30 mT, (H = f 3=0 298mT, kHz),f = 298 akHz), mild-temperature a mild-temperature rise up rise to up 315 to K, 315 the K,range the range at which at which the the elastin-like elastin- lipeptideske peptides collapse collapse provoked provoked the VEGF the VEGF release release in only in twoonly minutes two minutes after magnetic after magnetic stimulation stimulation without withoutsuﬀering suffering degradation degradation (see Figure (see 14 Figure). 14).

4.3.3.4.3.3. Thermal Thermal Stimulation Stimulation Additionally,Additionally, it it is is worth worth mentioning mentioning a a last last application application of of magnetic magnetic hyperthermia hyperthermia that that has has been been ableable to to produce DBS DBS (Deep (Deep brain brain stimulation) stimulation) at the at thenanometric nanometric level level in a mouse in a mouse with induced with induced thermal thermalsensitivity sensitivity in specific in specific neurons neurons selected selected by genetic by genetic technique. techniqu Thee. mouseThe mouse brain brain was injectedwas injected with withSPIONs SPIONs (around (around 22 nm) 22 andnm) exposed and exposed to an external to an external magnetic magnetic field which field provokedwhich provoked a local increasea local increasein temperature. in temperature With this. With procedure, this procedure, the selective the selective activation activation of the modified of the modified neurons wasneurons observed, was observed,without any without secondary any secondary effect in the effect neighboring in the neighboring neurons, whichneurons were, which preserved were preserved practically practically intact [97]. intact In[97 another]. recent study [98], the effect of magnetoelectric particles has been modeled to stimulate the brainIn another of an recent affected study Parkinson’s [98], the effect patient ofby magnetoelectric simulating a neural particles network has been with modeled electrical to stimulate levels like thethose brain of aof healthy an affected person. Parkinson The simulation’s patient estimatesby simulating optimal a neural values network for the with design electrical made from levels 20 like nm magnetite NPs, with a concentration of 3 106 under the action of an external magnetic field of 300 Oe those of a healthy person. The simulation× estimates optimal values for the design made from 20 nm magnetiteoperating NPs, at a frequency with a concentration 80 Hz. Undoubtedly, of 3 × 106 these under results the action open theof an door external to the magnetic effective developmentfield of 300 Oeof new operating innovative at a and frequency non-invasive 80 Hz. DBS Undoubtedly, methodologies. these results open the door to the effective development of new innovative and non-invasive DBS methodologies. 4.4. Magnetic Resonance Imaging 4.4. MagneticNuclear Resonance magnetic Imaging resonance (NMR) is a powerful magnetic phenomenon mainly used in clinics for imaging. In contrast to a surface-based sensor like GMR, NMR is capable of sampling an entire Nuclear magnetic resonance (NMR) is a powerful magnetic phenomenon mainly used in clinics volume in a non-invasive way. MRI can be combined with other diagnostic tools, like CT (X-ray for imaging. In contrast to a surface-based sensor like GMR, NMR is capable of sampling an entire computed tomography), PET (positron emission tomography) or US (ultrasound), to obtain more volume in a non-invasive way. MRI can be combined with other diagnostic tools, like CT (X-ray computed tomography), PET (positron emission tomography) or US (ultrasound), to obtain more

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accurate diagnosis, for which multimodal SPIONs can be afforded either combining several agents accurate diagnosis, for which multimodal SPIONs can be aﬀorded either combining several agents into a single carrier or by engineering a material which can be active in several modalities [99]. into a single carrier or by engineering a material which can be active in several modalities [99]. It is a known fact that the magnetic core creates a local magnetic inhomogeneity, which alters It is a known fact that the magnetic core creates a local magnetic inhomogeneity, which alters the relaxation time of hydrogen protons in the surrounding water molecules. This fact has motivated thedifferent relaxation studies time of based hydrogen on the protons use of in magnet the surroundingic NPs as water dual molecules.MRI contrast This agents fact has for motivated T1 and T2 di erent studies based on the use of magnetic NPs as dual MRI contrast agents for T1 and T2 relaxation, ﬀrelaxation, in order to avoid the conventionally used gadolinium chelates and their potential toxicity. in orderThe to avoidstrategies the conventionally developed so usedfar to gadoliniumoptimize the chelates magnetic and response their potential of magnetite toxicity. based NPs as MRIThe contrast strategies agents developed have so been far to focused optimize in the tailoring magnetic their response architec ofture: magnetite large based particles NPs containing as MRI contrastseveral agents SPM single have been cores focused (magnetic in tailoring beads), hollow their architecture: magnetite spheres large particles and single containing core NPs several of pure SPMmagnetite single cores or doped (magnetic with beads), transition hollow metals magnetite [100]. spheres and single core NPs of pure magnetite or doped withSPM transitionbeads, composed metals [ 100of dozens]. of SPIONs within a carbon shell [25] and with an approximate SPM beads, composed of dozens of SPIONs within a carbon shell [25] and with an diameter around 100 nm (see Figure 15), have been shown to present a large relaxivity (r2 = 218 mM−1 approximate diameter around 100 nm (see Figure 15), have been shown to present a large relaxivity s−1) combined with a negligible thermal rise when tested in magnetic hyperthermia tests (H = 30 mT, 1 1 (r2 f= = 218293 mMkHz)− . s− ) combined with a negligible thermal rise when tested in magnetic hyperthermia tests (H = 30 mT, f = 293 kHz).

80 m-Fe O @C 3 4 r = d(T -1)/d ) 60 2 2

-1

(s -1 -1 2 r =218,44 (mM-1 -1 s ) r22=218.44 (mM s )

1/T 40

(a) 20 -1 -1-1 -1 rr2=1634,1=1634.1(mM (mMs )s ) 2 0 1 2 3 0.000,00 0,050.05 0,100.10 0,150.15 0,200.20 0,250.25  (mM-1-1) Φ (mM ) (c)

(b)

Figure 15. (a) TEM image of multi-Fe3O4@C NPs, (b) scheme of the phantom NPs concentration distributionFigure 15. (b.1): (a) A TEM= 0.25 image mg/mL; of multi B = 0.1-Fe mg3O/4mL;@C C NPs= 0.05, (b) mg scheme/mL; D of= 0.025 the phantom mg/mL; E NPs= 0.005 concentration mg/mL; F =distribution0.0025 mg/mL (b.1):and A = G 0.25mg/mL;= 0 mg/mL; B = MR0.1mg/mL; T2-weighted C = 0.05mg/mL; image of D the = 0.025mg/mL; agar phantom E = (b.2);0.005mg/mL; MR T2*-weightedF = 0.0025mg/mL image and of the G = agar 0mg/mL; phantom MR (b.2), T2-weighted performed image at 9.4 of T the (BrukerBiospec) agar phantom (b.2); on a setMR ofT2*- agarwe phantomsighted image loaded of the with agar diff erent phantom(b.2), concentrations performed of NPs at (0.0025 9.4 T (BrukerBiospec) to 0.25 mM) and on (c ) a relaxivity set of agar calculatedphantoms from loaded T2 MRI with relaxation different on agar concentrations phantoms reprinted of NPs (0.0025with permission to 0.25 mM) from [and25], IEEE,(c) relaxivity 2016. calculated from T2 MRI relaxation on agar phantoms reprinted with permission from [25], IEEE, 2016. This combination of enhanced MRI contrast and poor thermomagnetic response offers a potential advantageThis for combination using these of materials enhanced in therapeuticMRI contrast situations, and poor like thermomagnetic brain applications, response where offer anys a temperaturepotential advantage increase is for highly using inadvisable these materials for patients in therapeutic [101]. In situations this case,, like despite brain the applications, high content where of magneticany temperature material content increase in is the highly NPs, inadvisable the carbon coatingfor patients shell [10 plays1]. In the this fundamental case, despite role the ofhigh thermal content insulation,of magnetic which material decouples content the magneticin the NPs, activity the ofcarbon the NPs coating from shell its thermal plays response.the fundamental role of thermalFurther insulation, enhancement which of decouples MRI contrast the hasmagnetic been achievedactivity of by the exploring NPs from the its e ffthermalect of shape response. on T2 relaxationFurther mechanisms, enhancement using singleof MRI core contrast magnetite has been NPs. achieved In this context, by exploring water-dispersible the effect polyethyleneof shape on T2 glycol-phospholipidrelaxation mechanisms (PEGphospholipid), using single coated core cubic magnetite SIONPs (seeNPs Figure. In this 16) showed context, extremely water-dispersible high r2 1 1 relaxivitypolyethylene (up to 761glycol mM-phos− s−pholipid)[102], yielding (PEGphospholipid) superior in vivo coatedMRI c details.ubic SIONPs Ascribing (see thisFigure exceptionally 16) showed highextremely relaxivity high to ther2 relaxivity magnetic (up shape to 761 anisotropy, mM−1 s−1) magnetite[102], yielding NPs superior with diff inerent vivo shapes MRI details. were studied, Ascribing this exceptionally high relaxivity to the magnetic shape anisotropy, magnetite NPs with different

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1 1 −1 −1 fromshapes octapod were studied, (D = 30 nm, from r octapod= 679.3 (D30 = mM30 nm,s r2 =)[ 610379.3] ± to 30 rod-like mM−1 s length−1) [103 of] to 30–70 rod- nmlike and length diameter of 30– 2 ± − − of70 4–12 nm and nm, diameter seeming toof conﬁrm4–12 nm, this seeming hypothesis. to confirm this hypothesis.

(a) (b)

FigureFigure16. 16. ((aa)) TEMTEM imageimage ofof coatedcoated cubiccubic SPIONsSPIONs andand ((bb)) sizesize dependentdependent r2r2 relaxivity,relaxivity,reprinted reprinted with with permissionpermission from from [ 102[102]] (ACS, (ACS, 2012). 2012).

Gadolinium,Gadolinium, a a paramagnetic paramagnetic ion ion with with the the largest largest number number of of unpaired unpaired electrons,electrons, isis the the most most eeffectiveﬀective T1 T1 contrast contrast agent, agent, which which has has however however the mainthe main drawback drawback of being of toxic.being Althoughtoxic. Although it is always it is usedalways in combinationused in combination with chelating with chelating molecules molecules with the aim with of the avoiding aim of its avoiding potential its risk, potential alternative risk, strategiesalternative are strategies seeking to are exploit seeking new to aspects exploit of new SPIONs aspects to produceof SPIONs versatile to produce dual contrastversatile agents, dual contrast taking ® advantageagents, taking of the advantage existing SPION of the (Ferucarbotran existing SPION (Resovist (Ferucarbotran), Bayer Healthcare) (Resovist® clinically), Bayer approved Healthcare) as MRIclinically contrast approved agent. as Ultra-small MRI contrast magnetite agent. NPsUltra with-small a diametermagnetite around NPs with 3.5 a nm diameter have been around studied 3.5 nm as candidateshave been forstudied dual-modal as candi MRIdates contrast for dual with-modal simultaneous MRI contrast T1/T2 switching,with simultaneous activity [104 T1/T2]. In switching this study,, the observed switching between T1 T2 contrast (see Figure 17) is ascribed to the fact that ultra-small activity [104]. In this study, the observed− switching between T1−T2 contrast (see Figure 17) is ascribed SPIONsto the fact are that single-dispersed ultra-small SPIONs in blood are andsingle produce-dispersed T1 contrast,in blood and while produce after their T1 contrast, extravasation while after and accumulationtheir extravasation in the and tumoral accumulation site, the NPs’ in the self-assembling tumoral site, the into NPs’ larger self clusters-assembling induces into the larger appearances clusters ofinduces T2 contrast the appearances modulated byof T2 magnetic contrast interactions. modulated by magnetic interactions.

Figure 17. T1- and T2-weighted MRI of a mouse bearing orthotopic 4T1 tumors before and after i.v. administrationFigure 17. T1- ofand ultra-small T2-weighted SPIONs MRI at of di aﬀ mouseerent time bearing points. orthotopic Bright contrast 4T1 tumors with before increasing and signal after i.v. in T1-weightedadministration MR of images ultra-small was observedSPIONs at in different the tumor, time particularly points. Bright peripheral contrast regions with increasing (inset), at signal early timein T1 points-weighted (i.e., 5–150MR images min) resultingwas observed from thein the single-dispersed tumor, particularly ultra-small peripheral SPIONs, regions whereas (inset), signals at early in severaltime points regions (i.e., of 5 the–150 tumor min) turning resulting dark from (arrow the single indicated)-dispersed were observed ultra-small in T2-weightedSPIONs, whereas images signals 24 h afterin several ultra-small regions SPIONs of the tumor injection turning because dark of (arrow ultra-small indicated) SPIONs were clustering observed in in the T2 tumor-weighted interstitials. images (Image24 h after reprinted ultra- withsmall permission SPIONs injection from [104 because], ACS, 2017). of ultra-small SPIONs clustering in the tumor interstitials. (Image reprinted with permission from [104], ACS, 2017).

Magnetochemistry 2020, 6, 4 22 of 27

4.5. Magnetic Prototyping of Functional Biological Structures One of the milestones in regenerative medicine is the fabrication of tissue, replacements or organs with biologically relevant functionality. This emerging field combines multiple strategies arising from different approaches like cell-based therapies, tissue-engineered biomaterials, scaffolds or implantable devices. The exposure of magnetized cells under a three-dimensional (3D) magnetic pattern, known as 3D magnetic bioprinting, allows the prototyping of organelles or autologous implantable biodevices without the need of a material substrate. Recently a 3D bio-fabrication system, by magnetic 3D bioprinting (M3DB), has been tested to generate innervated saliva secretory (SG) organoids using magnetized neural crest derived mesenchymal stem cells (human dental pulp stem cells (hDPSC). The magnetized cells were spatially arranged with magnet dots to generate 3D spheroids and afterwards cultured with fibroblast growth factor 10 to promote epithelial morphogenesis and neurogenesis. The preformed SG organoids were then transplanted into ex vivo model showing epithelial growth, innervation and production of salivary a-amylase upon FGF10 stimulation [105].

5. Conclusions Magnetite NPs with superparamagnetic behavior, commonly known as SPIONS, can be easily tailored in nanostructured materials combining functional organic or inorganic matrices (mesoporous or amorphous silica, biocompatible polymers like chitosan, k-carragenan, PNIPAM, PEG, PAA) to incorporate therapeutic or tagging moieties like drugs, aptamers or antibodies. The list of nanostructured materials containing magnetite NPs is immense, and the applications in nanomedicine cover almost any aspect from diagnosis, therapy or regenerative medicine, since in all cases the nanostructure inherits the intrinsic properties of magnetite that ensure multiple abilities like magnetic separation/detection/targeting, magnetofection, magnetic resonance contrast imaging, magnetic hyperthermia therapy and stimulated delivery, magnetic cell growth stimulation and 3D magnetic prototyping of organelles.

Author Contributions: Conceptualization: Y.P., S.Y.V.; writing: Y.P., M.G.G., L.d.C.A., A.A.P., P.G.A., R.S.G., J.P., C.T., S.Y.V., and J.R.; supervision and group direction: J.R. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the European Commission under (PANA project, Call H2020-NMP-2015-two-stage, Grant 686009; and MADIA project, Call H2020-ICT-2016-1, Grant 732678) and by the Consellería de Educación Program for Reference Research Groups project (GPC2017/015 and the Development of Strategic Grouping in Materials—AEMAT at the University of Santiago de Compostela under Grant No. ED431E2018/08, of the Xunta de Galicia. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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