Magnetic Iron Nanoparticles: Synthesis, Surface Enhancements, and Biological Challenges

Abstract

This review focuses on the role of magnetic nanoparticles (MNPs), their physicochemical properties, their potential applications, and their association with the consequent toxicological effects in complex biologic systems. These MNPs have generated an accelerated development and research movement in the last two decades. They are solving a large portion of problems in several industries, including cosmetics, pharmaceuticals, diagnostics, water remediation, photoelectronics, and information storage, to name a few. As a result, more MNPs are put into contact with biological organisms, including humans, via interacting with their cellular structures. This situation will require a deeper understanding of these particles’ full impact in interacting with complex biological systems, and even though extensive studies have been carried out on different biological systems discussing toxicology aspects of MNP systems used in biomedical applications, they give mixed and inconclusive results. Chemical agencies, such as the Registration, Evaluation, Authorization, and Restriction of Chemical substances (REACH) legislation for registration, evaluation, and authorization of substances and materials from the European Chemical Agency (ECHA), have held meetings to discuss the issue. However, nanomaterials (NMs) are being categorized by composition alone, ignoring the physicochemical properties and possible risks that their size, stability, crystallinity, and morphology could bring to health. Although several initiatives are being discussed around the world for the correct management and disposal of these materials, thanks to the extensive work of researchers everywhere addressing the issue of related biological impacts and concerns, and a new nanoethics and nanosafety branch to help clarify and bring together information about the impact of nanoparticles, more questions than answers have arisen regarding the behavior of MNPs with a wide range of effects in the same tissue. The generation of a consolidative framework of these biological behaviors is necessary to allow future applications to be manageable.

1. Introduction

Nanoparticles (NPs) and nanostructured materials have innovative properties due to their small size, large surface area, crystalline arrangement, composition, and reactivity [1,2]. These properties confer unique and specific effects, such as superparamagnetism, surface area and charge, and optic and magnetic properties different from their bulk presentation [3,4,5,6,7]. The design of these materials influences the final properties they present. Due to the different crystalline planes exposed on the surface of the said particles, they could externalize other atoms of the structure, which interact differently with the environment where they are found [8,9].

A well-known example is magnetic materials; when found on the nanoscale, iron oxide nanoparticles (IONPs) obtain a single-domain phenomenon known as superparamagnetism [10,11] that can shorten the relaxation times as contrast agents in magnetic resonance imaging (MRI) [12,13] travel to target locations with magnetic fields [11] and simultaneously play a role in both diagnostics and treatment, known as theranostics, by vibrating and heating. Recent design strategies of IONP-based nanoplatforms describe the rationale for the combination of other functional materials with IONPs and test their possible applications in smart nanomedicine theranostics [14]. However, dispersed NPs tend to form unstable suspensions in culture media, and modifying their surface chemistry, for example, with rhodium citrate, can increase their adverse effects, cytotoxicity, and uptake in breast cancer cell cultures [15]. Whether particles can become toxic by increasing their size (agglomerates) or by maintaining their nanometric size in dispersions is debated. Nevertheless, the increasing size implies a reduction in the number of atoms on the surface; thus, they become bulk materials and have reduced harmful effects [16,17,18,19,20,21,22].

Size is crucial when designing structures that interact at some point with the human body [23,24,25]. The range of cell sizes in the human body is wide, from the granules of the cerebellum in the brain (5 µm) up to the ovule (0.1 mm). Between these cells, a great variety of sizes are among the different systems responsible for maintaining homeostasis within the body. All these cells are three orders of magnitude larger than an NP [26]. Considering that one main application of magnetic nanoparticles is in diagnostics and treatment, such as imaging, the importance of these interactions becomes paramount to consent.

Imaging plays a crucial role in diagnosis because of the importance of the swiftness, efficiency, and accuracy of reliable early detection of diseases within the human body, such as cancer and inflammation [27]. Various contrast agents have been used to better visualize resolution by increasing the contrast between healthy and abnormal tissue that occurs within the human body, such as gadolinium, manganese, and barium [28]. Contrast agents for MRI are classified according to their specific characteristics, including: (i) chemical composition, (ii) route of administration, (iii) magnetic properties, (iv) effect on the magnetic resonance imaging, (v) biodistribution, and (vi) imaging applications (target/organ-specific agents) [29]. MNPs provide a potential promising alternative due to their improved magnetic characteristics (superparamagnetism) in terms of specificity, ease of functionalization, and biocompatibility.

Most of these agents are complexes of lanthanide elements such as Gd³⁺ or transition-metal manganese (Mn²⁺) with superparamagnetic magnetite particles [30]. These factors are used to shorten T1 or T2 relaxation times; the signal intensity on T1-weighted images is enhanced, or the signal intensity on T2-weighted images is decreased [31]. T1 and T2 are two different relaxation durations used to describe abnormalities in tissue. Most paramagnetic contrast agents are positive agents, which shorten the T1 relaxation time so that enhancements appear bright on T1-weighted images. However, these agents are used in an average of one-third of the analyses, given their low precision, problems brought on by the release of free gadolinium (Gd³⁺) [29], and the risk of presenting severe adverse effects in pediatric patients [32]. Dysprosium, superparamagnetic agents, and ferromagnetic agents are negative contrast agents [33,34], while enhancing parts appear darker on T2-weighted images [31]. MRI contrast agents incorporate chelating agents to reduce storage in the human body, improve excretion, and reduce adverse effects [35]. However, they must be used immediately due to their short shelf life [36].

Magnetite and maghemite are nowadays regarded as safe. They are now in clinical usage as MRI contrast agents [37,38,39], pharmaceutical agents [40,41], and a combination of both treatment and diagnostics, also known as theranostics [42,43,44], due to their significant superparamagnetic characteristics (around 40 to 90 Am²/kg) achievable because of their nanometric size (10nm) (Figure 1).

Magnetite and maghemite are the most suitable MNPs, but their iron oxidation states and structures differ, affecting their final physicochemical characteristics [45]. Other types of magnetic nanoparticles exist with similar promising properties, including titanomagnetite cobalt and nickel-based magnetic materials [46,47]. Nevertheless, the full impact of the nanoparticles in general and in the case of magnetic materials is still under study to correlate MNPs’ size, morphology, concentration, and crystallinity with different biological specimens. A collection of research from different cell lines and tissues has shown inconsistent results from cell–MNP interactions [48], such as uptake by mononuclear cell infiltration in mice liver [49,50,51,52], leaching of different ions such as Fe²⁺ [53,54,55], apoptosis via over-endocytosis, [56,57,58], oxidative stress by passive diffusion, receptor-mediated endocytosis unchaining mitochondrial stress [59,60], and DNA damage leaching Fe²⁺ into the cell’s core, increasing temperature while vibrating [61,62,63].

This array of properties that NMs possess have made it possible to generate significant advances in different areas, specifically biomedicine. For example, in the treatment and diagnosis of diseases, the role of MNPs has become increasingly important in the last two decades, but the short-, medium-, and long-term toxicological implications are set aside. This review focuses on the collection of various studies on assessment and challenges with regard to synthesis routes, biological evaluation, and the impact of iron MNPs to gather their different characteristics and properties, and studies presenting the possible effects in various biological assays and assessments to raise awareness of the correct use and disposal of MNPs to generate an integrative background of knowledge enabling improved visualization of the context behind the biological behavior of the iron MNPs.

2. Magnetic Nanoparticle Synthesis Methods

For manufacturing quality control and achieving the required outcomes for different applications, defined parameters for NPs’ diverse characteristics are essential. These NPs, composed of magnetic iron oxide, Fe₃O₄ (magnetite), and γ-Fe₂O₃ (maghemite), among other common structures with modified surface chemistry, are considered promising materials for widespread industrial applications in computer and sensing materials [64,65,66], the textile industry [67,68], ferrofluids [69,70], the remediation industry for the degradation of herbicides and emergent contaminants [71,72,73] and intrinsic magnetic properties [74,75], the biomedical area for iron deficiency anemia [76,77], imaging and diagnostics [78,79,80,81], heat cauterization treatments [82,83,84], and drug delivery [81,85,86,87,88] due to their chemical nature and biocompatibility. The unique characteristics that MNPs possess depend on the synthesis conditions.

Therefore, it is crucial to examine nanomaterials based on the physicochemical characteristics they acquired from the synthesis route used to obtain them (Figure 2).

Like most nanoparticles, MNPs have a high surface-area-to-volume ratio, implying they have qualities that are different from those of bulk material, such as a lower melting point, a lower sintering temperature, and distinct magnetic properties [35,89,90,91].

Different MNPs can be obtained by acquiring unique morphologies depending on the synthesis route and their conditions, such as temperature, pressure, reaction time, reagent casting, etc. (Figure 3), that could modify the MNPs’ physicochemical properties, surface functionalization, and magnetic properties. A ferromagnetic nanoparticle’s behavior is highly influenced by its size. When the size is reduced, it transforms from a multidomain particle to a single-magnetic-domain nanoparticle and finally to a superparamagnetic nanoparticle [81]. All the latter properties are achievable through different methods of synthesis to control the shape and coating in the case of functionalized materials.

Several synthesis routes have been created to obtain different morphologies, crystal growth, sizes, magnetic saturation, and surface charges of MNPs using parameters such as temperature, pressure, sonochemistry, and biological assistance with equipment, which can be simple to acquire, such as those obtained via an ultrasonic bath, to relatively complex microwave devices. These routes can provide specific MNP properties for different applications (

Table 1. Comparison between synthesis routes for the formation of MNPs *.

Synthesis Route		Temperature (°C)	Environment	Time	Size Control	Shape Control	Efficiency Output	Magnetite (XRD Pattern)	Ref.
Aqueous routes	Coprecipitation	<100	Insert atmosphere	Minutes	Relatively broad	Bad	High	Precursor-dependent	[92,93,94]
	Thermal decomposition	100–300	Insert atmosphere	Hours to days	Excellent	Excellent	High	Oxygen-dependent	[95,96,97]
	Hydrothermal	150–200	High pressure	Hours to days	Excellent	Excellent	High	Temperature-dependent	[98]
	Sol–gel	100–300	Ambient	Hours	Good	Good	Medium	Poor magnetite presence	[99,100,101,102]
	Microemulsion	<100	Ambient	Hours	Good	Good	Low	Poor magnetite presence	[103,104,105]
Assisted routes	Sonochemical assisted	<50	Ambient	Minutes	Good	Bad	Medium	Cavitation- and frequency-dependent	[106,107]
	Microwaved assisted	100–200	Ambient	Minutes to hours	Medium	Good	Medium	High magnetite presence	[108,109]
Biologic routes	Bacteria driven	Room temp.	Ambient	Hours to days	Broad	Bad	Low	Biologic-assistant-dependent	[110,111,112]
	Green	Room temp.	Ambient	Minutes	Relatively good	Good	Low	Medium magnetite presence. Leaves nature-dependent	[113,114,115]

* Table modified from [116].

).

2.1. Wet-Chemical Methods

2.1.1. Coprecipitation Synthesis

Coprecipitation is the ideal method for synthesis simplicity. This synthesis is a straightforward, well-known, cheap, and effortless process for obtaining MNPs (Fe₃O₄ and γ-Fe₂O₃) from a usual 1:2 molar ratio of ferric/ferrous salt solution by adding basic-pH solutions at standard temperature or increased temperature [117,118]. Most of the reactions could be helped with an inert gas atmosphere to prevent additional oxidation and to maintain their magnetic properties [119,120]. The ferric/ferrous ratio, the salt utilized, such as chlorides, sulfates, or nitrates, the reaction temperature, the pH value, and the ionic strength of the medium all influence the size, morphology, and composition of the magnetic particles [117], making the quality of magnetic particles entirely repeatable if the synthesis conditions are controlled. On the other hand, control over particle size attributes is reduced due to the rapid creation of particles. Overall, the coprecipitation methodology creates abundant MNPs, with strong magnetic properties (50 to 90 Am²kg⁻¹) but poor control over shape and size compared to other synthesis methods.

2.1.2. Thermal Decomposition

Thermal decomposition of organo-metallic precursor compounds in different solvents (organic and water) with high boiling points and stabilizing surfactants is a potential strategy for producing monodispersed magnetite nanoparticles without particle aggregation [83,121,122].

Thanks to the nucleation process, several synthesis variants have been developed for synthesizing MNPs with controllable size and morphology thanks to the nucleation process. Additionally, by promoting the adsorption of additives, e.g., the oleic acid–oleylamine ligand pair, the growth of nanocrystals can be achieved, inspired by the synthesis of high-quality semiconductor nanocrystals and oxides in a wet-chemical medium through thermal decomposition [95,97,121,123]. As a result, smaller superparamagnetic nanoparticles can be obtained. The nucleation condition of this method provides the possibility of the particles being separated from growth, unlike coprecipitation, preventing a more complex reaction process that can lead to oxidation [121]. In broad terms, the thermal decomposition synthesis process is one of the most appraised in terms of adequate size and morphological control. Thermal decomposition allows control over particle size, strong magnetic properties (40 to 90 Am²kg⁻¹), and high yield with easy access to precursors, with oxygen concentration being the crucial factor in producing the desired iron oxide structure.

2.1.3. Hydrothermal Synthesis

One of the most frequent ways of preparing NMs is hydrothermal synthesis. Straightforwardly, hydrothermal synthesis synthesizes single-crystal nanoparticles relying on the solubility of an aqueous solution (water) in the traditional crystal-making synthesis involving high temperatures and pressures [124]. It is essentially a solution-reaction-based synthesis method. The creation of NMs in hydrothermal synthesis can occur over a wide range of temperatures, from ambient temperature to extremely high temperatures (130–250 °C). The different vapor pressures in the reaction, in either low-pressure or high-pressure conditions (0 and 3–4 MPa), can be utilized to control the morphology of the materials to be synthesized, crystallizing the material in a sealed container [125,126] and highly improving the preferential growth of different planes of the MNPs [127]. Nevertheless, the high pressure and temperatures sacrifice the magnetic properties of the MNPs compared to other, cheaper, and simpler synthesis routes.

2.1.4. Sol–Gel Synthesis

In the materials science and engineering field, sol–gel synthesis is a traditional and commonly used technique in the wet-chemical approach, mainly used for NM fabrication (typically metal oxides) [101,102]. It usually begins with a colloidal solution that serves as a precursor for an integrated network of isolated particles. The system is shaped by a sol phase, a stable dispersion of colloidal particles in a solvent, and a gel, a continuous three-dimensional network that encloses the colloidal liquid phase. The aggregation of colloidal particles forms the network in a colloidal gel. The particles in a polymer gel have a polymeric sub-structure formed by the accumulation of sub-colloidal aggregates [116]. There are definite advantages of the sol–gel reaction over the previous wet-chemical or aqueous synthesis method, such as the simple dispersion in solvents, due to hydrophilic ligands on the surface of MNPs, which are used in the surface coating and for high control over size and shape; however, the high costs of the reagent precursors (i.e., alkoxides), the low yield compared to other techniques, such as coprecipitation, and poor obtention of magnetite structure limit the access of the sol–gel synthesis.

2.1.5. Microemulsion Synthesis

The self-assembly synthesis methodology of micro- and NMs has piqued interest in recent decades because of its unique features compared to bulk material analogs due to the mechanical and physicochemical property changes at the micro- and nanoscale. A surfactant stabilizes micro-domains in a thermodynamically stable and isotropic dispersion of two immiscible liquids (usually water and an oil-based compound) (Figure 4). It includes various structures and combinations that correlate to various self-assembled colloidal system performances [128]. The vital interaction to consider is the surfactant stabilizing the formed domains, which should be carefully chosen for the desired material. Iron oxide nanoparticles have been synthesized through this method. Iron ionic precursors, such as chlorides, in a basic-pH medium, such as ammonium, can be dissolved in water to create a reaction-ready space capable of producing the desired particle condensation [104,129,130]. This method offers control of the size of the nanoparticle through the solution of precursors and the available space provided in these solutions by adjusting the water-to-surfactant ratio, which entails a chemical reaction that converts soluble precursors to an insoluble in the aqueous phase, thus altering nanoparticle size [128,130]. Although monodispersed nanoparticles can be synthesized through microemulsion with reasonable control over size and shape, this technique has the lowest efficiency output, poor magnetite presence, and, consequently, low magnetic properties (20 to 60 Am²kg⁻¹).

2.2. Assisted Methods

2.2.1. Sonochemically Assisted

Acoustic cavitation is defined as the creation, expansion, and implosive collapse of bubbles in liquids bombarded with high-intensity sound, resulting in a transitory and localized temperature of 5000 K and a pressure of 1000 bar [85,131]. This collapse results in intense localized heat and pressures with minimal periods [132], commonly known as hotspots. This technology produces structures using the chemical effect of ultrasound. It provides a novel route without the use of high temperatures, high pressures, or extended reaction periods, ideal for the formation of materials with complex structures sensitive to extreme heat conditions (magnetic materials) [133,134]. The ultrasonic methodology route depends strongly on the cavitation and amplitude parameters applied to the particles. It can be observed sometimes as a complementary step in other synthesis routes to improve specific properties, such as coprecipitation, and to provide rapid reaction times or control of emulsion factors [132,133,135], producing the medium output of strong magnetic nanoparticles without the shape and size control of other techniques.

2.2.2. Microwave-Assisted

The orientation of molecules that occur when irradiated with any source of electromagnetic waves, aligning their dipoles within the external field, creates a strong internal heat resulting from the excitation with electromagnetic radiation [136,137]. The shape and size of the obtained nanoparticles, as well as their magnetic characteristics, may vary slightly depending on the experimental parameters, such as the precursor material or reaction temperature [109,136,138,139,140,141]. Microwave heating of a solution of iron oxide nanostructures such as magnetite and hematite has been used to improve their magnetic properties, sacrificing crystal structure [138,142]. This process has a variety of advantages over conventional heating-based procedures, such as high reaction rates and excellent product yields, due to the rapid localized heating provided by microwaves [143,144], reducing process time and energy costs compared to the conventional thermal methods, making microwave-assisted synthesis methods more appealing for iron oxide nanoparticles.

Both approaches are relatively simple processes in time and reaction times, obtaining specific control over the nanoparticles.

2.3. Biological Synthesis Routes

Several researchers are now looking at creating dependable, clean, and environmentally acceptable experimental procedures for NP synthesis. The biosynthesis of NPs utilizing various biological components, such as plant derivatives, is a strategy with a lot of promise.

Oxidation and other thermic methods tend to sacrifice structure to gain control over the synthesis, size, magnetic properties, morphology, etc. [18,88,141,145]. Several types of magnetotactic bacteria can create biologically manufactured domain-specific iron oxide nanoparticles called magnetosomes via biomineralization, which can be caused by Fe(III)-reducing bacteria such Thermoanaerobacter, Bdellovibrionota, Proteobacteria (alpha, eta, zeta, gamma), SAR324, and Phoenix dactylifera species [146,147,148,149], typically grown from a ferric oxyhydroxide precursor at 65 °C. The resulting magnetite crystals, 5–140 nm in size, are confined in a biological membrane regulating the magnetosome’s size and form [148,150,151]. Magnetosomes in bacteria are grouped in chains with the [111] crystal plane with narrow down-size distribution [152].

Green synthesis of NMs can result from, for instance, bacteria-driven synthesis of living organisms’ capacity to reduce metal and oxides into defined structures within their metabolism. Additionally, different plant species have biological extracts or the capability to hyper-accumulate metal ions in tissues to form nanoparticles or decrease them in various organs to generate metal oxide structures, as shown through different phytoremediation studies [153]. As a result, many extracts from other plant parts, such as the leaves, stem, root, fruit, and seed, have been employed to synthesize nanoparticles. Using natural extracts of plant parts is a very cost-effective and environmentally friendly approach. It does not require the use of any intermediate base groups such as silane (Si-H), mercaptoundecanoic acid (MUA), oleic acid, cetyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP) [154], or sodium cholate, among others [155]. Likewise, expensive equipment and precursors are not required. Because of the variety of metabolites in the plant extracts, such as phenolic compounds that have ion-reducing capabilities, most of the efforts associated with obtaining MNPs using plant extracts have been through the implementation of the leaves as the source of these extracts [156,157,158,159]. Several aspects must be considered when producing NMs through leaf extractions: the solvent, the plant origin, extraction methods, reaction time, and external industrial chemical reagents [155,160].

MNPs are typically functionalized with various substances (such as citrate, phosphate, chitosan, polyethylene glycol, albumin, silica, iron oxides, and metals) to increase colloidal stability and biocompatibility since uncoated MNPs have the propensity to flocculate when exposed to physiological fluids [161] and tend to present unfavorable effects [162]. The assessment of the size and zeta potential as a function of time in various media (culture medium, phosphate buffer, in the absence or presence of fetal bovine serum (FBS)) will help us to understand how MNPs behave when they are in contact with bio-relevant culture media. Although it is a novel approach to NP production, excessive energy consumption, long reaction times, and the difficulty of obtaining precise chemical reactions to describe the mechanisms underlying the biosynthesis process are the primary issues with the synthesis process [163,164].

2.4. Surface Coating

Just like bulk materials, iron materials are prone to rust and corrosion. Iron oxide nanoparticles cannot be used as a core material to produce treatments or pharmacological agents per se without the addition of functional [82,165], biocompatible [166], inert, or target coatings [167,168]. MNP design is essential in biomedical applications. The features of the MNP core are determined by form and size, but the choice of added ligands and coatings significantly impacts the colloidal stability and functions [169], further specifying their properties for later applications.

It is a known fact that MNPs have an inherent instability when preserved over long periods due to their magnetism [170,171,172], which leads to a loss in dispersibility because of the aggregation and formation of large particles and reduces the surface energy, generating a loss of magnetism. Non-coated iron nanoparticles easily oxidize when are exposed to air due to their high chemical activity and unstable spinel structure, especially magnetite and maghemite nanoparticles. They have a maximum shelf life of about 8 to 12 months [116,173]. As a result, chemically stabilizing simple iron oxide nanoparticles to prevent damage during or after their subsequent exposition and/or application becomes an essential factor to consider because most biological organisms and applications require aqueous solutions to be administered [44,174]. Generally speaking, polymers, SiO₂, carbon-derived structures, and metal material coats of a nanoscale substance constitute a family of composite nanoparticles [175] to reduce their negative impacts on living organisms and serve as ligands for functionalization [167].

2.4.1. Silica Coating

Due to its chemical stability, diversity in surface alterations, and limited loss of initial magnetism, silica (SiO₂) has been regarded as the best material to safeguard MNPs. Silica is beneficial in attaching different biological ligands to the iron oxide surface. Silica coatings, metal, or metal oxide coatings help stabilize the nanoparticles in the solution. Silica coating is the preferred material when applying layers to MNPs due to its increased biocompatibility, ability to bond with organic linkers, and simple synthesis methodology [176,177]. Different methodologies for coating MNPs and nanoparticles are based on the desired properties, thickness, silica percentage, and later functionalization [100]. The Stöber methodology is the most used and straightforward because of its simple application. It requires a sol–gel precursor, which is hydrolyzed and condensed to obtain SiO₂ sheets that will cover the desired material [178]. A modified microemulsion method using a multilayer silica coating applied to nanoparticles with relative simplicity, assisted by microwave radiation, has proven very effective at controlling single layers of a few atoms [179].

2.4.2. Carbon-Based Coatings

By adding functional groups to the iron oxide surface, such as hydroxyl (-OH), carboxyl (-COOH), amino (-NH₂), or silane (-SH), various bioactive compounds can be bonded. Due to the benefits of linking several kinds of molecules to the coating [174,180], carbon coatings are popular. These provide a magnetic core material with strong chemical and thermal stability, high intrinsic electric conductivity, and an efficient oxidation barrier. Hydrophilic carbon coatings also provide enhanced stability and dispersibility. With the co-precipitation and surface oxidation of CNTs, nanoparticles have been effectively created, resulting in hybrid materials with improved conductive properties from 16 meV at 20 °C to a maximum of 67 meV at 120 °C [181]. Different synthesis methods can be used to produce core–shell nanostructures. The three-step process uses MNPs as precursors, applying any synthesis route previously mentioned, e.g., heating or applying plasma, to prepare the surface (which may cause oxidation), coat it with polymers, and finally, carbonate it with an annealing treatment [182,183,184].

2.4.3. Metallic Coatings

One of the disadvantages, if not the most important, of MNPs is the tendency to change the crystal structure of the magnetite via oxidation [185]. The idea of linking a nanoscale oxide phase with a metal surface to create a metal–oxide hybrid material with unique features is appealing. Monodispersed iron oxide–metal nanostructures with binary characteristics, such as core–shell, core–satellites, or dumbbell structures, achieve enhanced stability and biocompatibility through the change in charge [186]. Noble metals in bulk appear to have inert behavior, biocompatibility, and no significant interactions with other elements, which could make them ideal in biomedical applications [187]. Due to their interaction with external fields and the ease with which they are recovered, reused, or altered with magnetic manipulation, MNPs, namely magnetite and maghemite, are particularly appealing as components of multifunctional NMs, and the general biocompatibility of noble metal nanoparticles makes these materials the ideal combination for biomedical applications [186,188].

Conventionally, forming an amorphous or oxidate phase in nanostructured magnetic materials has been a drawback of the synthesis process. Given its often negligible contribution to effective saturation magnetization, the amorphous fraction is frequently portrayed as a “dead” magnetic component and a material waste [189]. The core–shell morphology with an ordered core and a shell with structural/magnetic disorder often describes this scenario as the simplest example of MNPs.

2.4.4. Polymer Coatings

Microemulsion polymerization and the sol–gel technique are frequently used in situ synthesis methods that traditionally produce a core–shell or matrix-dispersed structure [49]. It is still difficult to regulate the colloidal stability and thickness of the created structures. Post-synthesis methods such as the one-pot approach, self-assembly, or heterogeneous polymerization are commonly chosen for the polymeric coating of MNPs to avoid excess particle oxidation [190,191].

Utilizing polymer coatings has allowed for increased MNP stability and an expansion of the application space by altering different functional groups [192] to provide properties such as the connection with optoelectronic segments, imaging, diagnosis, and treatment, enabling conjugated polymers with a delocalized electrical structure [193].

These are brief applications of covers and functionalization coatings that can be applied to MNPs, known as multicoated or multicore. However, more properties can be attached to these materials, combining coating types [194,195] to solve deficiencies of the iron NP core, such as biocompatibility or recovery of the material. A typical method for creating multicore nanoparticles is to trap many magnetic cores inside a polymeric matrix [195]. Three main approaches to the synthesis of polymer/SPION hybrid multicore particles have been reported in the literature: (i) polymerization in the presence of the nanoparticles [196]; (ii) SPION formation from iron salts along an existing polymer particle; and (iii) in the emulsification process (ESE) within a precipitating polymer [197].

2.5. Nanocomposites

Nanocomposites are materials with more than two phases, which have units with at least one dimension within the nanoscale present in the matrix material to produce integrated functional systems with additional properties [198]. Nanosized particles of various magnetic materials have been inserted into extended matrix materials (i.e., organic or inorganic polymers). Additionally, the future importance of magnetic nanocomposite materials made from renewable resources will undoubtedly rise to safely recover the material, making it a reusable source [199,200,201]. They blend the characteristics of the filler with the matrix material to create innovative functional materials that are tailored to the requirements of a specific application, such as when magnetite nanoparticles are enclosed in a short-chain amphiphilic block copolymer combined with an organic pigment during the self-assembly of higher-order polymer structures to create biosynthetic, bifunctional, and magnetically active materials with poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) [202].

In general, a wide variety of host materials, such as organic polymers, silica, or even liquid media, are used in nanocomposites [203,204,205].

3. Biological Challenges

Nanotechnology, NMs, and nanostructures have been around humans since the early days at the beginning of the industry and early metallurgy [206,207,208]. We can state that the human–nanostructures interaction is not contemporary. Although the appropriate determination of “nano” was given centuries later by Richard Zsigmondy, the Nobel Laureate in Chemistry (1925), he was the first to suggest the term “nanometer” [209] to the scientific world. He was the first to measure the size of particles such as gold colloids [210,211,212], and he developed the term nanometer to describe particle size using a microscope. However, only about 30 years ago, the significance of these nanostructured materials was apparent to researchers of disciplines as diverse as medicine and photoelectronics due to the different and novel characteristics conferred by their size. Researchers have discovered that size affects materials’ physicochemical characteristics, such as their magnetic properties [119,213,214].

MNPs can intervene in different steps and applications in both medical and industrial sectors. Because these particles are composed of iron and oxygen, they have been targeted as the standard material for medical applications because of their magnetic properties. They can act as a carrier for drug and heat delivery [81,86,215,216], a potential treatment when undergoing vibrating or carrier structure breaking processes [82,217,218,219,220], and even a contrast and targeting agent [80,213,219,220,221], shortening periods between the diagnosis and treatment, which is a crucial factor in medical processes and biocompatible needs. At least, that was the original thought. Over the past two decades, research has shown mixed results from the same tissue in different assays assessing the toxicity of MNPs (

Table 2. Different biological assays in MNPs *.

Tissue	Concentration	Morphology	Size	Coating	Methodology	Effect	Ref.
Fibroblast (hTERT human)	0.1–0.02 mg/mL	Spherical	7.8–9.6 nm	Dextran, albumin Lactoferrin, ceruloplasmin	BrdU assay	Cellular death	[222,223,224]
Lung cells (A549)	20–40 mg/kg	Spherical	20–107.7 nm	Bare	TB staining, ROS, Comet	Enhancement of free radicals and reduction in the GSH DNA oxidative injuries (Comet) Low—no toxicity (TB, ROS) Increased TP and LDH Non-biomechanical damage Cell Young’s modulus decreased (25–28%)	[225,226]
Liver rat cell (BAL3A rat)	20–40 mg/kg 25, 50, 75, 150, 300 µg/g	Spherical	107.7 nm 20–30 nm	Bare Liposomes PEG	MTT, LDH	Nontoxic below 75 µgmL−1 Nonalcoholic fatty liver disease (NAFLD) inflammation LDH leaking Iron overload affected by NAFLD	[226,227,228]
Mesenchymal mother cell (MSC human)	50, 100, 250 mg/mL 25, 50, 100, 150 µg/mL	Spherical	80–150 nm 48 nm	Protamine sulfate PDA	Comet	No significant effect Increased proliferation index and migration ability	[229,230,231]
Kidney cells (Cos-7 monkey)	15 mg/kg 1–100 µg/mL	Spheric-like Ferrofluid	13–122 nm 9.7 nm	Phospholipid-based polymeric micelles DOX	MTT, MTS	Cell viability reduced Particle charge (+)-induced high cytotoxicity Oxidative stress, reverted by tissue	[232,233,234]
Macrophage (human)	2.73 mg/mL	Ferrofluid Ellipsoidal	320–490 nm	Bare SiO2	MTS, BrdU assay	Time-dependent cell viability (7 days, 20%) Induced M1 activation	[235,236]
Nervous system cells (human, PC12)	0–1000 µg/mL	Spherical Hollow spheres	5–100 nm	PGA SiO2 Dextran Bare	MTS, LDH	MTS increased production DNA fragmentation, apoptotic Conformational changes in Tau protein Oxidative stress	[237,238,239]
Endothelial cells (BAECs, HUVECs)	50 µg/mL 0–100 µg/mL 0, 300, 600 µg/mL	Spherical Spherical–like	50–600 nm 5–10 nm 10 nm	Bare Dextran PSC	PI staining, Redox	Cellular intake Cell viability 80 ± 3% Promotes cell survival by autophagy Peroxidase-like activity	[240,241,242]
Cancer (multiple)	50, 100, 500 µg/mL	Spherical–like	6 nm	DMSA	MTT	High cell viability >90% Trigger immune response High ROS activity	[240,241,242,243]
Vascular system (A10 rat)	50, 100, 200, 400 µg/mL	Spherical–like	150–160 nm	Bare Citric acid	Redox, MTT	Decreased cell viability Increased actin and calponin expression Concentration-dependent toxicity Migration of EPC reduced	[242,244,245]

* Table modified from [246]. BrdU—bromodeoxyuridine, TB—trypan blue, ROS—reactive oxygen species, GSH—glutathione, TP—total protein, LDH—lactate dehydrogenase, MTT—3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide, MTS—3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, PI—propidium iodide, PEG—polyethylene glycol, PDA—polydopamine, DOX—doxorubicin, PSC—polyglucose sorbitol carboxymethyl ether, DMSA—dimercaptosuccinic acid.

). However, the concentration range is extensive. Most research focuses on the spherical shape of the nanoparticle, giving controversial results depending on the study. Spherical particles produce no effect when analyzed through ROS and TB in lung cells, but a comet assay reveals oxidative DNA damage, as shown in Table 2.

Although many studies have assessed the biological effects of MNPs, a consensus has not been achieved on safety parameters.

Biological processes imply a series of pathways, reactions, and signaling at a molecular level, all taking place around 1 to 100 nm, which is, coincidently, the action size range of nanostructured materials. It is unsurprising then that, as such, nanotechnology and nanostructure materials require special attention and safety parameters to function and not interfere with any other healthy cells and systems when applied to biological systems (Figure 5).

Like any type of active drug substance and new material, nanostructured materials must be subjected to different test methods to determine their effects and their impact on health, regardless of whether they will be used in the medical sector or not [17,247,248,249]. Among the most used biological assays are the in vivo, in vitro, and the so-called ex vivo tests. For the ex vivo tests, a target organ (heart, lungs, kidney, liver) is kept in a controlled system outside the body to observe its interactions with an active ingredient (such as nanoparticles or any vasoactive controls, acetylcholine, phenylephrine) in real time [250,251,252,253]. Ex vivo tests have obvious advantages over other methods, including rapid results, a relatively low cost, and reduced use of live animal exposure [254,255].

In the case of heavy metals, such as bulk iron, adverse effects at concentrations around 350 µgdL⁻¹ are observed [256]. Additionally, the leaching of different ionic species can occur in the ferrous state (Fe²⁺), which is insoluble, or in the ferric state (Fe³⁺), which can directly interfere with agents related to oxidation [54,55,257]. Hemoglobin as molecular oxygen (O₂) and nitric oxide (NO₂, NO₃), a representative mediator in cardiac muscle [258], is a free radical synthesized enzymatically by endothelial NO synthase (eNOS), neuronal synthases (nNOS), and inducible (iNOS) [258,259,260,261].

Detection of the non-favorable response of different substances in biological systems can be classified into three main categories, depending on the biological section (cell, tissue, organ, and system) that has been tested. In vitro is specifically for single cells and tissue sections in controlled conditions in culture. In in vivo tests, the complex interactions between systems (cardiovascular, respiratory, endocrine, digestive, renal, and nervous) are tested. Moreover, a relatively new biological evaluation test has been raised, ex vivo, which maintains isolated organs or segments in an external, controlled environment to detect changes in physiology in real time (Figure 6).

In vivo studies can evaluate the primary stages of the adverse effects due to their availability and relative ease of use compared to other studies. Ex vivo tests are a relatively new approach to toxicology, bringing advantages over in vitro studies because of their capacity to assess real-time effects on specific organs or tissue sections (heart, lungs, kidneys, liver), isolating the tissue from the general bloodstream. Ex vivo analysis tends to isolate specific organs from the animal and maintain them in a controlled environment. Complex organs such as the heart require a more delicate approach in retrieval from the bloodstream, necessitating a constant flow rate or pressure modes [262]; however, isolation from blocking of the aorta and immediate recovery and attachment to the external parameters can preserve the organ long enough to study it [250,253,263,264]. Lastly, in vivo assays are the last evaluation before human trials and determine the complex response of different biological system interactions and the impact of active substances.

3.1. In Vitro Toxicology

Different in vitro tests are available to verify toxicity; the lactate dehydrogenases test (LDH), sulforhodamine B (SRB) assay, protein assay, neutral red assay, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay are a few of the often-used assays [59].

Tetrazolium salt, derived from MTT, is transformed into a purple, insoluble formazan complex by a mitochondrial dehydrogenase enzyme. These enzymes are only found in active mitochondria; hence, the process only occurs in healthy cells [265]. The LDH leakage test, commonly used due to its dependability, speed, and ease of assessment, determines lactate dehydrogenase leakage to the extracellular medium in damaged cells [266]. Trypan blue is the test most often employed for viability studies. The test provides an uncomplicated way to assess cellular viability by fixing the cells in a solution of trypan blue and paraformaldehyde after being deposited onto slides. Nonviable cells have a heavy blue stain, but viable cells do not [266]. However, this method does not distinguish between apoptotic cells.

The safety assessment of MNPs on cell lines (in vitro) is uncomplicated, straightforward, and affordable since the experiment can be reliably controlled. Toxicity in MNPs has been associated with dosage dependence, time, and surface modification [267].

Since in vitro testing is the first approach to understanding the impact of material in a living organism, it is not unusual that the information collected from in vitro and in vivo tests happens to be controversial. This results from the complex bodily interactions and different systems coexisting in an in vivo test [268]. However, these tests unify the results of the full impact of the materials or pharmaceutical agents.

3.2. Ex Vivo Toxicity

Physiological models have been used for a long time as a valuable tool for the study of endogenous molecules such as hormones, neurotransmitters, and other mediators under normal or pathophysiological conditions, such as cardiovascular, respiratory, and digestive disorders, to name a few [250,252].

Physiological ex vivo models of isolated tissues and organs allow the evaluation of the particular functioning of a tissue or organ in NPs’ presence, for example, ducts related to the cardiovascular system (aorta, arterioles, mesenteric, etc.), respiratory system (trachea, bronchi, bronchioles), and digestive system (small or large intestine); or in the study of organs such as the heart, kidney, lung, and liver; and the biochemical communications between the organs and tissues [269]. By separating the organ from the general circulation, identifying metabolites generated from the interaction with MNPs becomes easier [270].

This is a novel approach to assessing the toxicology of MNPs, but the road ahead is still long and full of opportunity. These assays produce exciting results in cardiovascular heart contractility and NO production.

3.3. In Vivo Toxicity

Although in vivo toxicity investigations take longer, cost more, entail ethical considerations, and have more complicated techniques (such as toxicokinetic processes) [271], they offer a clear benefit over in vitro experiments. Even though there is not enough in vivo research on the toxic effects of MNPs, those studies that do exist provide valuable information on possible toxicity, such as alternating current biosusceptometry (ACB) [272]. The ACB system is a biomagnetic approach previously described and used in pharmacological and gastrointestinal research in animals and people [273], as well as recently for detecting MNPs [274].

For the best results in the therapeutic or diagnostic application, the MNPs are aggregated in a specific tissue using a magnet, which might result in high concentrations in that location [275]. This may result in large amounts of free Fe ions in the exposed tissue, resulting in cellular damage and/or significantly influencing subsequent cell generations [276].

When MNPs enter the body, they interact with the bodily fluids and then bind to the proteins to form a protein corona, a nanoparticle–protein complex. The MNPs–corona complex is essential for the particles to perform as intended because MNPs interact with one another in vivo through the formation of this structure, which serves as the biological identity of the MNPs [277]. As a result, understanding the surrounding protein composition, particularly the individual proteins present in the MNPs–corona complex and their affinities, is crucial for understanding how the MNPs will act in vivo.

These toxicological assays and approaches provide a different view of a material’s effect on other parts of complex systems, increasing the understanding of its full impact on nature and living organisms. All these efforts have expanded the body of knowledge on nanotoxicology and help in regulating the novel properties that nanoscale materials bring to different fields (

Table 3. Biological assessment of treatments for MNPs.

Biological Studies	Type of Assay	Assays in MNPs	Ref.
In vitro	Suspension (HL60, K562) Monolayers (MCF-7, U87MG) Cultured	CCK8, MTT, TB, LDH, Comet	[268,278]
Ex vivo	Langendorff isolated system, in silico studies	Perfusion pressure, protein expression, mediator count, liver, spleen, lungs, heart	[250,264,279]
In vivo	Biodistribution, histological staining	VIP, liver, spleen, lungs, heart	[268,280]

CCK8—cell counting, VIP—vacuum filtration process.

).

4. Regulation and Control

Even though research and toxicity approaches have been made to identify the potential toxicity of MNPs, there are no clear regulatory parameters to control these materials’ production, interaction, and disposal. Different organizations have made several proposals since 2011 around the world (

Table 4. Definitions of nanomaterials of different global organizations.

Organization	Nanomaterial Definition *	Status	Last Meeting/Proposal	Ref.
NANoREG (European Union, EU, European Commission, EC)	Taken from EC: “Any intentionally produced material that has one or more dimensions of the order of 100 nm or less or that is composed of discrete functional parts, either internally or at the surface, many of which have one or more dimensions of the order of 100 nm or less”.	Toxicological data gathering	2014, updated by NanoFATE in 2022	[281,282]
International Organization for Standardization (ISO)	“Any material with any external dimension in the nanoscale or having an internal structure or surface in the nanoscale”.	Terms and vocabulary for nano-objects	2017	[283]
FDA (United States of America, National Nanotechnology Initiative, NNI)	Taken from the NNI: “The understanding and control of matter at dimensions between approximately 1 and 100 nanometers, where unique phenomena enable novel applications”.	Nonbinding recommendations for manufacturers	2014	[284,285]
ECHA (European Union)	“A natural, incidental, or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in size range 1 nm–100 nm”.	Guidance for terms, vocabulary, and sample dispersion and aggregation of nanoforms	Draft 2021	[286]
CEPA (Canada)	“Any manufactured substance or product, as well as any component material, ingredient, device, or structure, if it has at least one external dimension that is at or within the nanoscale, or if it has internal or surface structure that is at the nanoscale, or if it has all dimensions that are smaller or larger than the nanoscale and exhibits at least one nanoscale property or phenomenon”.	Guidance framework for adapting nanomaterials to existing practices	Draft 2022	[287]

* The definition was extracted literally from each organization manuscript cited in the reference column.

).

The idea of safe-by-design is becoming an increasingly significant approach to nanosafety. Nanotechnology-based products must be safe to use throughout their lifespan, from manufacture to disposal, recycling, and reuse. A well-established general idea in industrial innovation, safe-by-design was first developed for nanomaterials in the European Union’s (EU) flagship project NANoREG [281].

The legislation on Registration, Evaluation, Authorization, and Restriction of Chemicals, or REACH, in the European Union is responsible for regulating chemical agents [288]. REACH defines any NMs as “nanoforms,” creating a new guideline with which to contemplate these new materials and their properties. It has a section establishing that a material composed of less than 50% nanostructures is classified as a bulk material without considering its dissemination to the environment or biological organisms. However, these guidelines were defined in 2013, and just recently, the term nanoform was implemented [286].

Another administration agency, the U.S. Food and Drug Administration (FDA), which is in charge of the regulation of product safety, has provided a guideline to specify if a manufactured good possesses nanotechnology or nanoscale properties with two criteria: (i) whether a substance or finished product is designed to have at least one exterior dimension, an interior structure, or a surface structure in the nanoscale range (approximately 1 nm to 100 nm) and (ii) whether a substance or finished product is developed to show characteristics or phenomena, such as physical or chemical characteristics or biological consequences, even if these dimensions fall beyond the nanoscale range, up to one micrometer (1000 nm) [285]. The FDA guideline, however, was released in 2014 and concluded that nanomaterials are not subject to premarket review but urged the industry to consult with the agency early in the product development process, leaving nanomaterials stranded with no regulations.

Canada’s regulations on environmental and health hazards are controlled by the Canadian Environmental Protection Act (CEPA), which put out regulatory parameters in 2013 on how to modify current risk assessment procedures considering the unique features shown by chemicals at the nanoscale in line with the Organization for Economic Co-operation and Development (OECD) proposal [287]. In the latest draft of June 2022, the CEPA proposed mediating NM risk in three appraisals: (i) substance identity, taking into account composition, shape, agglomeration, surface chemistry, dissolution in biological media, and any other specification that the material may possess according to the manufacturer; (ii) exposure assessment, be it direct contact or indirect contact (water, air, soil), and release potential or fragmentation; (iii) hazard assessment, considering toxicological endpoints such as acute toxicity, repeated-dose toxicity, genetic toxicity, reproductive or developmental toxicity, carcinogenicity, toxicokinetics, etc. The final step is the risk characterization, as determined by the points established in part 5, Section 64 of CEPA: (a) the material has or may have an immediate or long-term harmful effect on the environment or its biological diversity; (b) the material constitutes or may constitute a danger to the environment on which life depends; and (c) the material constitutes or may constitute a danger in Canada to human life or health.

It is well-known that MNPs have different properties than their counterparts on a macroscopic scale. Studies have been conducted to determine their toxicity in various body organs with multiple materials [289,290,291]. However, reported viability results on the same material in different tissues within the body have presented contradictory results depending on the type of tissue to which these materials were administrated [1,51,59,219,248,292,293,294], so their regulation must be specialized. Furthermore, it is marked correctly to prevent a significant effect on society and the environment [295].

5. Conclusions and Perspective

MNPs exhibit interesting novel properties due to their superparamagnetism, reduced size (10 nm), and versatility in forming functionalized composites that increase their applications as theranostics. However, these same problem-solving qualities can be responsible for causing possible adverse biological impacts in the future if they result in excessive and unregulated use. Different global initiatives have been launched to address this new branch called nanotoxicology and nanoethics. This must be considered when applying any NMs in any sector, not just MNPs, whether it is industry or medicine, to ensure the correct handling of the material and prevent other environmental and health problems due to the improper use of new versatile materials. However, the vast properties of NPs, even with the same composition, be it size, morphology, surface charge, magnetism, or general reactivity, present a problematic task ahead in mitigating NPs’ effects. Slowly but steadily, though, the regulations for MNPs are taking shape. Still, the interaction and pathways of NPs are yet to be uncovered. There is no comprehensive image of the toxicological impact of magnetic nanoparticles on biological cells. Even though the literature is full of extensive analyses of different biological systems discussing the toxicology aspects of MNPs used in biomedical applications, they give mixed and inconclusive results depending on the assay implemented to determine their biological effects with an exhaustive list of parameters. Narrowing this down to a consensus list would help to generate a consolidative framework for these biological behaviors to allow manageable future applications. Finally, using only one nanomaterial parameter in the same tissue to make a consensus on the adverse effects and impact of MNPs leads to unreliable results.