Progress in magnetic Fe₃O₄ nanomaterials in magnetic resonance imaging

2.1 T ₂ sign imaging of Fe₃O₄ nanomaterials

Ferrites are crystalline iron oxides whose magnetic properties were recognized for their commercial importance as early as 1933 [20]. Ferrites have the general formula Fe³⁺ ₂O₃·M²⁺O, where M is a divalent metal ion such as manganese, nickel, iron, cobalt, or magnesium. Magnetite is a naturally occurring ferrite in which the metal ion (M) is a ferrous iron (Fe²⁺). More importantly, the outermost layer of iron atom contains five electrons similar to gadolinium, which can change the relaxation time of protons and engender greater r ₁ and r ₂ relaxation effects, thus showing certain potential in MRI applications [21]. So far, there are three kinds of iron CAs using the T ₂ sign imaging on the market, Feridex (r ₂ = 120 mM⁻¹ s⁻¹), Resovist (r ₂ = 186 mM⁻¹ s⁻¹), and Combidex (r ₂ = 65 mM⁻¹ s⁻¹), respectively [22]. The following is in introduction to the current T ₂ CA study.

The stability of CAs is one of the key problems affecting the application of CA [23]. Researchers have tried various ways in which to improve the stability without changing the magnetic imaging performance. Among them, the copolymerization of an organic polymer is a relatively direct and an effective method [24]. There are two preparation methods available: (i) one is to first synthesize nanoparticles and then to modify the coating with an organic polymer, and (ii) one-pot method by thermal precipitation copolymerization method [25] (Figure 1).

Figure 1

Preparation methods of iron CAs.

Certainly, there are other ways, for example, Kluenker et al. [26] presented a solution phase seed mediated synthesis of iron oxide superparticles colloidal with flower- and hedgehog-like morphologies starting at dispersible spherical maghemite and nanoplate hematite (HEX) templates to overcome the drawbacks of particle assemblies for functional nanodevices such as low mechanical stability, lack of interfacial electronic communication, and poor processability [27]. Changes in solvent, type of iron salts, and temperatures have been used to synthesize different types of nanomaterials (Figure 2; the NPs are mostly spherical and faceted, and an average particle size is 18–20 nm). As for the higher effective radius and anisotropy and inhomogeneity of the particle-generated magnetic field, both longitudinal and transversal relaxation sign effects show obvious enhancement (r ₁ and r ₂ values are 0.093 and 1.774 mM⁻¹ s⁻¹ for HEX SPs). The r ₂ relaxivity could be increased by a factor 2.5–64.36 mM⁻¹ s⁻¹ showing the potential for application as a T ₂ CA. Generally, the viability of the cells decreases with increasing Fe concentration and reaches a minimum of 70–80% at 100 μg/mL NPs, which exhibits less virulence. Certainly, this preparation method also provides a new way for industrial production.

Figure 2

Flow diagram of solvent planting process [26].

Early organic polymer coating with dextran, polyethylene glycol (PEG) [28], polyvinyl alcohol, and chitosan as an outer cladding has been reported [29]. These polymers can effectively improve the water solubility and stability of nanomaterials, but the size of nanoparticles is also significantly increased, relevant studies were shown below.

Hong et al. [30] coated SPIONs with dextran to stabilize monocrystalline structures to form a stable magnetic fluid, so the stability and biological properties of dextran-coated SPIONs were significantly improved, but its application was limited by the larger particle size. Mailander et al. [31] modified Resovist (generic name: ferucarbotran) with carboxydextran in Feridex (ferumoxides) and original dextran. Many studies have shown that the PEG coating of iron oxide nanoparticles (IONPs) improves their stability (with a mean diameter (S _d) of 10 nm) [32]. Although pure PEG can improve the stability of IONPs, it will soon be removed by the RES system. Therefore, PEG can act as remedy for such defects. For example, Xie et al. [33] stabilized oleic acid/oleylamine coated NPs by PEG-dopamine, which demonstrated a remarkable abatement in nonspecific uptake by macrophage cells. Amstad et al. [34] proved the better stability of PEG compounds bound to iron oxide NPs coated by nitrocatechols compared to that with catechols. In recent years, research on other types of polyorganisms has gradually increased, such as amino acids and phosphate polymers [35].

At present, the clinical contrast medium should not only be stable enough and non-cytotoxic, but also meet the requirements of low dose, a certain degree of targeting, and so on [36]. Therefore, it is imperative to improve its magnetic properties while achieving targeted properties. Monoclonal antibodies (MAbs) could distinguish malignant tumors from normal tissue and could be used for selective targeting [37]. Kubovcikova et al. [38] coated ferric material with poly-lysine (MFPLL, S _d = 85 nm, and a hydrodynamic diameter (H _d) of 119.2 nm) to improve the stability in aqueous solution, and then conjugated a selected specific antibody VII/20 (Ab-MFPLL, with a S _d of 128 nm, and a H _d of 170.1 nm), which was capable of internalization and targeting to recognize the overexpression of carbonic anhydrase IX (CAIX) in tumors. Stability studies show that Ab-MFPLL and MFPLL are stable in acidic conditions up to pH = 6 and alkaline medium up to pH = 8, respectively. Simultaneously, MFPLL showed a notable enlargement in H _d after 44 h at room temperature, and the Ab-MFPLL showed no changes throughout the 74 h experiment, proving their excellent stability. The relaxivity values r ₂ and r ₁ of MFPLL were 487.94 and 1.81 mM⁻¹ s⁻¹, respectively, and the ratio r ₂/r ₁ equaled to 270, which proved the potential as T ₂ CA [39]. Furthermore, Ab-MFPLL samples preserve the property of magnetic hyperthermia differing from other USPIOs [40]. The specific absorption rate values of MFPLL were 14–15 W g⁻¹ at a frequency of 190 kHz and field strength of c. 8 kA m⁻¹, which evinces the potential for the application of magnetic hyperthermia. Yin et al. [41] used targeted peptide WSGPGVWGASVK (peptide-WSG) conjugated Fe₃O₄ NPs coated with dextran (SPIONs@Dex-WSG), to meet practical requirements. Preparation of SPIONs@Dex-WSG relies on use of sodium citrate as an intermediate material to connect WSG and SPIONs@Dex. The average size of SPIONPs@Dex-WSG (60.66 nm) decreased compared with SPIONPs@Dex (165.20 nm) because SPIONs@Dex-WSG can significantly improve the agglomeration of nanoparticles, avoiding their elimination by RES, and prolonging blood circulation time. Cytotoxicity experiments showed that SPIONs@Dex-WSG was only slightly toxic to SKOV-3 uterine cancer cells and non-toxic to normal cells. Simultaneously, Prussian blue staining indicates that only SKOV-3 uterine cancer cells appeared blue while normal cells were not stained, which indicated that SKOV-3 uterine cancer cells had good targeting and a weak inhibition rate. Its plasma half-life was as high as 10.6 h, which also provided support for its enrichment in tumors. The VSM and T ₂ for SPIONs@Dex-WSG were 44.65 emu g⁻¹ and 229.70 mM⁻¹ s⁻¹, respectively.

The outer layer is either dextran or amino acid, and its bonding mode is nothing more than a carbon–oxygen bond or a carbon–nitrogen bond [42]. When the external environment changes, it is easy to break the structure of nanoparticles and release SPIONs, resulting in reduced stability. Demin et al. [43] formed a stable P–O–Fe bond by the coordination of phosphate on PMIDA (1:1, mol/mol) with the iron of MNPs (PMIDA@Fe₃O₄ NPs) and obtained an optimum condition for the modification of MNPs at reaction at 40°C for 3.5 h; at the same time, PMIDA exhibits selectivity recognizing for HER-2 receptors in MCF-7 tumor cells [44]. PMIDA@Fe₃O₄ NPs had good colloidal stability (concentration = 0.1–2.8 mg/mL, pH = 2–11) and high magnetic properties (with values r ₂ and r ₁ were 341 and 102 mM⁻¹ s⁻¹, respectively). PMIDA@Fe₃O₄ NPs as a T ₂ contrast agent was demonstrated in the first time for liver studies in vivo (dose of 0.6 mg kg⁻¹).

Although the organic outer polymer cladding layer can improve the stability of IOSNPs, it is the low magnetic sensitivity that still limits their further application [45]. The ability of MNPs was affected with shape and composition engineering. Yin and colleagues [46] doped dysprosium (the magnetic moment of Dy (10.6 µ _B) was 3.5 times larger than Fe) into Fe₃O₄ nanoparticles and coated it with polyethylene alcohol on its outermost layer to improve the sensitivity of IOSNPs. The r ₂ value was 123.2 s⁻¹ mM⁻¹, which was nearly double than the pure IOSNPs (67.8 s⁻¹ mM⁻¹) and substantially surpassing that of Feridex and Resivist [47]. More importantly, low dose doping did not cause a toxic reaction, but in vivo imaging was yet to be reported. Yin and colleagues only considered the composition of the nanoparticles, without considering the influence of the shape of them. Gao et al. [48] not only considered the configuration of nanoparticles, but also studied the effect of the different metal doping ratio on the properties of nanoparticles. They manufactured zinc ferrite octapods (Zn _x Fe_3–x O₄) with different Zn ratios, and studied an effect of the different ratios of Zn in Zn _x Fe_3−x O₄, indicating that Zn _x Fe_3−x O₄ (x = 0.44) with octahedral structure owns a notable M _s of 89.0 emu/g and r ₂ value of 989.1 mM⁻¹ s⁻¹. The specific production steps are as follows: FeCl₃ was reacted with sodium oleate in a mixed solution of ethanol and distilled water (v/v = 1:1) for 4 h, filtered after cooling, and the red oil layer was collected and concentrated, which contains iron oleate. Zinc oleate was used in the same way (temperature increased to 100°C for 0.5 h after dissolving the metal salt, and then impurities removed). Under the protection of N₂, Zn _x Fe_3−x O₄ octapods were realized by calcination at 350°C for 2 h, where x represents the ratio of iron oleate to zinc oleate. The sensitive detection can be even 0.7 mM lower for orthotopic and metastatic hepatic tumors (1/10 for the clinical dose), which demonstrated the potential as a T ₂ CA [49].

Similarly, nanomaterials mixed with other metals can improve contrast performance and have favored attentions interest recently [50]. Luminomagnetic nanoparticles that possessed core/shell structures (and Fe₃O₄ NPs coated with luminescent material) show some advantages [51]. Although optical imaging and magnetic imaging can be achieved because of the existence of rare earth up-conversion fluorescent nanoparticles, the imaging effect is not ideal, especially magnetic imaging [52].

Karthi et al. [53] doped Nd³⁺ to fluorapatite (FAP) and coated Fe₃O₄ nanoparticles by a hydrothermal method. The specific preparation method is as follows: adding Nd³⁺ droplets to Fe₃O₄ nanoparticles coated with FAP, while maintaining an alkaline environment and calcining at 600°C for 1 h, the final product was obtained (FFN, in rod shaped with average length 40 nm). It exhibited a superparamagnetic property (M _s of FFN is 1.3 emu/g at 1.6 mT) and showed excellent optical properties (emission at 1,060 nm). Cytotoxicity results indicated that when the concentration reached 500 μg/mL, the L929 cell survival rate remained up to 84%, showing good biocompatibility, but the real imaging effect was not discussed.

Whether using doped metal or researching the outer layer, it is necessary to improve the performance of CAs and advance their clinical application. As an iron agent for the T ₂ signal, such research is extensive, and its biological applications likewise [54]. To further improve the biological performance of CAs, Chandra and colleagues [55] modified amino acid (aa) into ferrite to prepare the magnetic fluid (Fe³⁺:Fe²⁺:aa = 1:2:3, particle size about 15 nm) for the first time. It can be used to image stem cells because of the coating of amino acid, so the biological toxicity of magnetic fluid is trivial. The theoretical basis is that amino acids can be used in vaccine research and development, and ideal biocompatibility makes it stable in the body; what is more interesting is that MRI is applied to MSCs imaging for the first time [56]. The imaging effect showed that its T ₂ imaging effect was better than that of pure ferrite and commercial magnetic fluid, showing the amino acid-modified magnetic fluid’s relative advantage for T ₂ MRI in MSCs.

Similarly, Li and colleagues [57] have also used ferrite for its magnetic properties and MRI performance to prepare a special purpose ferrite coated with CPG (forming a lecithin-like structure) and anti-CD205. Targeted CD205 and bionic CPG were used to transport ferrite to lymph nodes to achieve real-time monitoring of lymph nodes. The results showed that T ₂ imaging of lymph nodes was superior to its performance in other organs and proves that ferrite has been effectively transported to the lymph nodes.

VEGF-A is a cytokine overexpressed in benign or metastatic cancer cells, such as breast, ovarian, cervical, lymphoma, and colon, as well as in other cell lines such as epithelial cells [58]. Ma et al. [59] coated oleic acid–Fe₃O₄ and triphenylamine-divinylanthracene-dicyano (TAC) with poly(l-lactic-coglycolic acid) by O/W method. Simultaneous modified anti-VEGF antibodies were used to form anti-VEGF/OAFe₃O₄/triphenylamine-divinylanthracene-dicyano@poly(l-lactic-co-glycolic acid) NPs. This system was capable of recognizing overexpressed VEGF-A at as low as 68 pg/mL in different cell lines. The r ₂ reached 86.2 mM⁻¹ s⁻¹, and the magnetic resonance signal appeared within 30 min by intravenous injection and reached the peak at 60 min in tumors, which proved its good selectivity. At the same time, the optical properties of the TAC can be used to achieve photothermal therapy while imaging, to achieve the integration of diagnosis and treatment.

The instability of CAs in colloid and the difficulty in drug release limit its clinical application in vivo [60]. To solve the problems of unstable micelles in vivo and difficulty in drug release, Yang et al. [61] used organic thiol-based silicon as the outermost layer of micelles. Specific steps are as follows: (i) based on the self-assembly characteristics of polycaprolactone-block-poly(glutamic acid) (PCL-b-PGA) in aqueous solution, Fe₃O₄ nanoparticles and doxorubicin (Dox) were initially coated as the core; (ii) a thiol-containing silicone layer was formed on the outer layer with the help of 3-mercaptopropyltrimethoxysilane to form biodegradable nanoparticles (Fe₃O₄/Dox NPs); and (iii) PEG was modified on the outer layer of Fe₃O₄/Dox NPs (FDPOMs, with S _d of 120 nm). The M _s of FDPOMs was 7.4 emu g⁻¹, which was lower than the pure Fe₃O₄ NPs (33.2 emu g⁻¹) because of the outermost organic layer and organosilica components surrounding the Fe₃O₄ nanoparticles [62]. The r ₂ value of FDPOMs was 192.06 mM⁻¹ s⁻¹ in vivo; interestingly, the tumor region showed the strongest T ₂ sign after injecting FDPOMs for 4 h, which demonstrates that the FDPOMs own potential as T ₂ CA. The outermost layer contains sulfhydryl, so it can be degraded by glutathione, thus releasing drugs in the tumors, making the tumor inhibitory 7.9 times higher than that of Dox alone [63], however, due to the presence of Dox, the biological toxicity is high, when the adriamycin content exceeded 25 μg/mL, the cell survival rate was less than 50%.

2.2 T ₁ sign imaging of Fe₃O₄ nanomaterials

Defects of T ₂ signal imaging are gradually exposed, the dark view produced by transverse relaxation easily causes confusion in diagnosis, and strong magnetic distance causes “bioboming effect” generating an interference magnetic area [64]. T ₁ signal imaging is a bright field of vision produced by longitudinal relaxation, which can overcome the defects of T ₂ signal application [65].

When reducing the effective particle size of the nanoparticles, the value of surface area/volume ratio is effectively increased, causing accumulation; in addition, the outermost layer of iron atoms contains five lone pairs of electrons, which can generate a high T ₁ [66]. Based on this idea, Wei et al. [67] studied zwitterion-coated-ultra-SPIONs (ZES-SPIONs, the S _d was 4 nm). Although ZES-SPIONs demonstrated excellent T ₁ imaging potential and biocompatibility, the preparation process is complicated and polluting, especially the sophisticated phase transition process, and therefore its practical application is limited.

Hao et al. [68] applied zwitterion to improve the preparation method. The improved preparation has two innovations: (1) preparing amphiphilic dopamine sulfonate (ZDS) as the outer layer to improve the application of dopamine; and (2) using diethylene glycol as solution and adopting two-step method to obtain Fe₃O₄ NPs coated with ZDS (ZUIONs). The size of ZUIONs was only 3.3 nm and the H _d was 7.0 nm; more importantly, it showed perfect stability, and above all, the program only needed one pot. The nanoparticles improve the T ₁ signal (r ₂ = 2.4 mM⁻¹ s⁻¹ and an r ₂/r ₁ = 2.2 at 1.0 T); meanwhile, it is exciting that it has showed enough cycle time without any immune response and metabolism. To sum up, it shows great clinical application value, but only as a T ₁ CA.

Li et al. [69] also developed biodegradable Fe₃O₄-coated catecholate amphiphilic block to improve imaging effect and stability, but the complicated preparation is limited for its application as it requires multi-step reactions to guarantee the amphipathy. Miao et al. [70] applied α-amino acid N-thiocarboxyanhydrides (NTAs) with polysarcosine (PSar) to synthesize poly-3,4-dihydroxy-l-phenylalanine-b-polysarcosine (PDOPA-b-PSar) via controlled ring-opening polymerizations, and then PDOPA-b-PSar coated Fe₃O₄ NPs to get micellar nanoparticles of Fe³⁺@PDOPA-b-PSar, which simplifies the process of production, including phenol hydroxyl protection and deprotection and so on, so as to facilitate future industrial production. Fe³⁺@PDOPA-b-PSar not only shows the higher ability of T ₁ MRI (3.0 T, 20°C) but also exhibits a longer circulation time (2.5 h) compared to commercial Gd³⁺ and DTPA-Gd³⁺. Simultaneously, their biocompatibility is excellent when evaluated in NIH 3T3 cells, but the release and metabolism of Fe³⁺ in vivo and absence of T ₂ signal MRI were not considered by the authors.

The composition of the outer cladding and the length of the chain layer will affect the magnetic properties and biological properties of the final nanoparticle [71]. Fortin et al. [72] used poly[oligo(ethylene oxide) monomethyl ether methacrylate] (POEOMA) covalently connected with either poly(methacrylic acid) (PMAA) as an anchoring block to increase blood retention time [73]; at the same time, brushed-PEG chains were applied to ensure the branching state (straight chains become brushed chains, P2). The preparation process was designed to replace chain PEG with brushed-PEG: because of the presence of a large number of –COOH radicals, the intermolecular force was greater and the particle size is smaller, but the zeta charge was reduced to −1. There was no obvious precipitation to prove the stability of –COOH radicals in 72 h of blood compatibility experiment. In vitro T ₁ imaging for P2 showed that the values of r ₁, r ₂, and r ₂/r ₁ were 5.3 mM⁻¹ s⁻¹, 20.7 mM⁻¹ s⁻¹, and 3.8, respectively, which were significantly lower than chain-like outsourcing, but higher than commercially available Supravist SHU-555C [74]. In vivo angiography showed that there was a strong signal at 2 h (blood half-lives 2–5 h), which was mainly related to the presence of more –COOH in the outer layer.

The magnetic properties are independent of the type of external coating and are widely studied. At the same time, the phenomenon of ferroptosis therapy (FT) appeared in people’s vision, conferring a huge advantage in the diagnosis and treatment, and thus received much attention [75].

Since Dixon et al. first proposed FT in 2012 [76], it has been found that the FT efficacy was low because the high concentration of iron in the treatment process was demanded (75 mg iron/kg mice, the principle is shown in Figure 3) and FT therapy has not been used in the treatment of brain tumors, which is that metallic nanoparticles are toxic to the central nervous system and difficult to penetrate the blood–brain barrier (BBB) [77].

Figure 3

The principle of FT for killing cancer cells.

To effectively cross the BBB, Shen et al. [78] prepared Fe₃O₄/Gd₂O₃ NPs loaded cisplatin (CDDP) and modified lactoferrin (LF) and RGD dimer (RGD2) (FeGd-HN@Pt@LF/RGD2) to improve FT efficacy by synchro raising the topo-reactants’ concentrations (Fe²⁺, Fe³⁺, and H₂O₂) in tumor cells, and to cross the BBB and reach the tumor cells by LF receptor, RGD2 receptor, and their small size (6.6 nm). The FT mechanism is as follows: when entering the brain, Fe²⁺, Fe³⁺, and CDDP can be released from the nanoparticles after endocytosis, NADPH oxidases can be activated by CDDP to produce H₂O₂, and all reactants work together to generate reactive oxygen species, causing brain cancer death. These nanoparticles are a potential T ₁ CA as for the high r ₁ value (56.57 mM⁻¹ s⁻¹) and low r ₂/r ₁ (1.25) at 1.5 T, which can be ascribed to the special morphological results and ease of access to water. Furthermore, the cure rate for the tumor mice was nearly 100% after 18 days treatment, which exhibits an excellent FT effect.

The FT effect has a good therapeutic effect and a certain degree of targeting, and certain clinical application potential. Of course, there are other applications, such as in liposomes and vesicles. Madhuri et al. [79] cleverly coated the inner layer with polymersome and Gd nanoparticles, and simultaneously targeted the surface folate and the chemotherapeutic drug methotrexate on the surface modification to reform the magnetopolymersome (MPS). When achieving the target, the chemotherapeutic drug was released, so as to achieve the purpose of simultaneous effect of chemotherapy. Gold nanoparticles for targeted dual-mode therapy and imaging have been used. MPS has high biocompatibility because of its identical structural analogues with the cell membrane and also achieves high drug loading and targeting as well as development imaging. The r ₁ and r ₂ of prepared AuNFs@MPS were 60.57 and 200.0 mM⁻¹ s⁻¹, respectively (r ₂/r ₁ = 3.3), which proved that AuNFs@MPS was potential as T ₁ MRI CA [80]. Although it can realize multi-mode treatment and diagnosis, the production process is cumbersome and is not suitable for further development.

3.1 Application of Fe₃O₄ in bimodal imaging

Reducing the particle size of the nanoparticles not only produces a higher T ₁ signal, but also controls the particle size to generate a simultaneous T ₂ signal for dual-modulus imaging. The preparation method is mostly based on thermal decomposition [82], as shown below.

Since 2005, Chen et al. [83] used dodecanethiol-polymethacrylic acid (DDT-PMAA) to prepare 1–4 nm single-layer gold nanoparticles, providing theoretical support for preparation of ultra-small-MNPs. Cooper et al. [84] used this principle to first synthesize the ligand of trithiol-terminated poly(methacrylic acid) (PMAA-PTTM), which was then used to synthesize ultra-small magnetic Fe₃O₄ nanoparticles, but it did not apply it for MRI. Who is really applied those nanoparticles to MRI is Li et al. [85], who prepared monodispersed hydrophilic ultra-small Fe₃O₄ NPs (UMIONs, D = 3.3 ± 0.5 nm) for detailed MRI imaging performance studies. The results showed that their longitudinal relaxivity value at 4.7 T (r ₁ = 8.3 mM⁻¹ s⁻¹) was nearly twice as much as Gd-DTPA (r ₁ = 4.8 mM⁻¹ s⁻¹) and the transversal relaxivity value (r ₂ = 35.1 mM⁻¹ s⁻¹) was six times that of Gd-DTPA (r ₂ = 5.3 mM⁻¹ s⁻¹). The results showed that the liver and kidneys signal for T ₁ and T ₂ values improved by at least 26% and up to 70% in vivo, which is stronger than Gd-DTPA at the same dose. Majeed et al. [86] also synthesized UMIONs (D = 4.6 ± 0.7 nm) under high magnetization (50 emu g⁻¹) using the water-soluble ligand DDT-PMAA, and modified Dox on the outer layer to achieve the purpose of magnetic targeting therapy; however, this study only discusses cytotoxicity, loading capacity, and magnetism, and does not mention magnetic imaging performance.

There are other methods, for example, Sarlak et al. [87] first synthesized Fe₃O₄ NPs (10 nm) by thermal precipitation method, and then coated hydrophilic cellulose-poly citric acid to obtain the final product (CNC-PCA/Fe₃O₄). The most interesting thing is that the hydrated particle size after coating does not increase, and it also decreases (13.2 to 12 nm), which proves that there is no precipitation after coating, resulting in the high M _s (52.2 emu g⁻¹), so as to produce high r ₁ (13.8 mM⁻¹ s⁻¹), r ₂ (96.2 mM⁻¹ s⁻¹), and a considerable r ₂/r ₁ (7.0) at 3.0 T.

Although thermal decomposition can achieve small-scale production (typically 5 g), it does not meet the huge clinical demand; therefore, achieving large-scale and stable production of USIONs has become one of the key preconditions for its clinical application [88], whereas other methods cannot achieve large-scale production because of the constraints of particle size stability and shape constancy [89]. Starsich et al. [90] used flame aerosol technology to prepare different quantities of silica coatings with different proportions of iron(iii)acetylacetonate and hexamethyldisiloxane, and focused on structure–function relationships and cytocompatibility. This mode of production of USIONs can achieve stable, uniform, and constant production. The advantage of those prepared USIONs is that iron oxide exists in the form of monomer and its particle size is about 1.5 nm. It has good imaging effect as T ₁ contract agent, however, because of silica coating causes a poor T ₂ signal, the lethality of the four kinds of cells in the study of configuration and biocompatibility is not more than 15% even when the iron content reaches 1 mg/mL, which proves its potential for application in T ₁ signal analysis.

Although the particles coated with ferroferric oxide alone can achieve dual-modulus imaging, their sensitivity cannot meet clinical requirements [91]; therefore, the development of highly sensitive CAs to meet the clinical needs has become one of the burning issues to be solved.

3.2 Application of Fe₃O₄ cladding metal in the outer layer of nanoparticles

Generally, neither Gd-based coordination complexes on the market today nor iron oxide or manganese metals can satisfy the high accuracy of diagnosis in tumor treatment [92]. The main reason is that other highly magnetic metals in the outer layer of nanoparticles can improve the sensitivity of nanoparticles, which can meet the requirements of high-precision imaging in clinical application [93].

Yang et al. [94] relied on this theory to prepare the late-model nanoparticles with cladding metal in the outer layer. The preparation process was as follows: first, polyethylenimine (PEI) coated on the synthesized of Fe₃O₄ NPs surface, then Gd(acac)₃ forms the outermost layer at a suitable distance in the outer layer of Fe₃O₄@Gd₂O₃ NPs (YFGN) with an interstitial-hollow space forming a yolk-like structure, which ensures the formation of an ideal T ₂ signal due to water molecules can pass through the porous shell and approach Fe₃O₄ nanoparticles in the core [95]. Besides, the special yolk-like structure owned a large specific surface area and mesopore, so that it can be used as a drug delivery system [96], so cisplatin was adsorbed to PEI by the special yolk-like structure. In addition, tumor cells in a slightly environment can produce more protons to replace platinum chelates, so as to achieve the effect of pH response, so cisplatin was released from YFGN through the special yolk-like structure. Final aminating PEG-COOH and folic acid improve the hydrophilicity and achieve the purpose of targeting, thus achieving the goal of complex (FA-PYFGN-CDDP). At 15 min post-injection, responding signal-to-noise ratio (ΔSNR) was 88.53% and 41.71% for T ₁ and T ₂, respectively, which testified quality targeting and imaging performance for FA-PYFGN. Concurrently, the r ₁ and r ₂ values of PYFGN were 7.91 and 386.5 mM⁻¹ s⁻¹, respectively (r ₁ value of Gd²⁺ was 4.8 mM⁻¹ s⁻¹, and the r ₂ value of original Fe₃O₄ was 268.1 mM⁻¹ s⁻¹); however, Gd²⁺ remained in other organs, especially in the kidneys. FA-PYFGN-CDDP showed advanced targeted ability and reduce the side effects of CDDP [97], and the improvement of therapeutic effect was nearly 1.5-fold; however, the retention of gadolinium in kidney and the tedious preparation process limited its practical application.

To effectively reduce the side effects of gadolinium retention, Xiong et al. [98] prepared Fe₃O₄ nanoparticles and coated with MnO₂ to improve diagnostic accuracy and decrease aggregation in the reticuloendothelial system and its toxicity. In the acidic condition of tumors, Mn²⁺ was released and a T ₁ signal was produced, which reduced aggregation of nanoparticles in normal tissues; at the same time, the inner Fe₃O₄ produced a T ₂ signal, so that the CA with a double signal (in pH response terms) could be achieved. In addition, the stability of Fe₃O₄ coated with carbon is significantly improved, and carbon in the application can make the nanoparticles have other excellent properties in the later research, such as thermotherapy and fluorescence imaging (FL) because carbon possesses various excellent properties of chemically stable, tunable bandgap, good thermal conductivity, and stability [99].

The preparation process is as follows: the carbon shell coated Fe₃O₄ (Fe₃O₄@C, called FOC) nanoparticles were prepared by hydrothermal calcination of ferrocene and hydrogen peroxide at 240°C, and the supernatant was discarded under a magnetic field. Then the oxidative KMnO₄ was added; Fe₃O₄@C@MnO₂ (FOCMO, with S _d of 130 nm) nanoparticles were prepared through the oxidation–reduction reaction [100] among the reductive FOC and the oxidative KMnO₄. The r ₁ was 5.33 mM⁻¹ s⁻¹, and r ₂ = 364.20 mM⁻¹ s⁻¹ at pH = 5.0 in vivo (r ₁ = 3.56 mM⁻¹ s⁻¹, r ₂ = 396.57 mM⁻¹ s⁻¹ at pH = 6.5), which suggested sufficient sensitivity to acidic environment. After intravenous administration of FOCMONPs, T ₁ value was enhanced for 127% at 24 h. Furthermore, T ₂ value decreased 71% in vitro. In addition, the cell viability exhibited even a high (>85%) concentration up to 200 ppm for 24 h with HeLa and 4T₁ cells, which demonstrated that FOCMO NPs can improve accuracy diagnosis.

3.3 Application of metal-doping-Fe₃O₄ in bimodal imaging

Metal-doping-Fe₃O₄ nanoparticles can be divided into two categories: one is that changing the iron valence to form new nanoparticles, and the other is that doping alone does not change the iron valence.

3.3.2 Metal-doping-Fe₃O₄

3.3.2.1 Application of Fe₃O₄ doping other metals in the outer layer

The effective doping of other metals in the outer layer can effectively improve the sensitivity of CA [105]. The dumbbell-hybrid nanotrimers (DB-HNTs) showed dual contrasts such as gold-doping-Fe₃O₄ NPs [106]; however, the preparation process was arduous and complicated. On this basis, Gong et al. [107] covered a manganese ferrite (MnFe₃O₄) nanoparticles enhanced dual models effect to overcome the tedious process of preparation, but Mn²⁺ was latent toxicity to human body. Similarly, Zhou et al. [108] showed Gd-embedded-Fe₃O₄ NPs as dual MR contrasts; however, the Gd²⁺ could lead to kidney malfunction [109]. Park et al. [110] also reported uranium-doped Fe₃O₄ nanoparticles (EuIO) as the novel agents. EuIO were synthesized by thermal decomposition method. The outer layer was modified by citric acid (EuIO-Cit). EuIO-Cit produced a higher r ₁ value (17.2 mM⁻¹ s⁻¹, r _1Gd-DTPA = 4.8 mM⁻¹ s⁻¹) with a smaller r ₂/r ₁ value (5.2) as well as a high hydrophilicity and smaller size (5 nm), which showed better dual modulus development potential. The same magnetic properties are superior, but there remains the issue of the toxicity of foreign elements.

To solve the side effects caused by doping other metals, Thapa et al. [111] verified nitrodopamine-PEG coated single core truncated cubic Fe₃O₄ NPs (ND-PEG-tNCIOs, the average edge length was 12 nm) to enhance imaging effect. The result showed longitudinal and transverse relaxivity of 32 ± 1.29 mM⁻¹ s⁻¹ and 791 ± 38.39 mM⁻¹ s⁻¹ at 1.41 T at 30°C, respectively. As nitrodopamine-PEG owned the excellent biocompatibility, it could effectively avoid phagocytosis of the immune system so as to increase the iron content of the target local, thus improving the accuracy of T ₂ imaging [112], at the same time, the inner structure of the cube makes it easier to aggregate at the tumors than a spherical structure, which makes the imaging results more accurate [113].

3.3.2.2 Application of Fe₃O₄ doping other metals in the inner core

Although embedding T ₁ contrast metal (Gd³⁺, Eu³⁺, Ni²⁺, or Dy²⁺) into Fe₃O₄ NPs could not only improve the sensitivity of contrast medium, but also provide dual-mode imaging as for the fact that the internal impaction ensured the same magnetic field direction and increases mutually [114], as shown in Figure 5, rare-earth contrast materials occasionally cause nephrogenous systemic fibrosis [77]. Therefore, doping some metals as necessary elements of the body becomes an effective means.

Figure 5

The influence of doping other metals in different positions on magnetic field strength: (a) outer layer; (b) inner layer [115].

Based on this theory, Lu et al. [115] embedded Mn²⁺ into Fe₃O₄ NPs with hydroxyl-PEG-phosphonic acid through a one-pot reaction (Fe₃O₄/MnO, with a size of 20 nm) to improve synergistically imaging performance with safe and accuracy because Mn²⁺ was T ₁-weighted CA [116]. The stability test indicated that the nanoparticles retained a single scattering peak even though their size increased to 35 nm from 21 nm after 21 days in water. The r ₂ and r ₁ values were 209.6 ± 0.7 and 22.8 ± 0.3 mM⁻¹ s⁻¹ (v. [Fe + Mn]), respectively, which was over five times than commercial Magnevist (4.4 ± 0.3 mM⁻¹ s⁻¹) in terms of the r ₁ value and were higher than pure Fe₃O₄ NPs and MnO NPs in terms of the r ₂ and r ₁. Furthermore, the r ₂/r ₁ ratio was 9.2, which suggested its potential as dual-mode CA [117]. The results of T ₁ and T ₂ in vivo were similar: 5 min after injection, clear images of two kinds of signals could be seen in lung cancer, and 1 h post-injection, the signals could be seen in liver, which indicated that the dual-modulus could be clearly presented and maintained for sufficient time. Despite the excellent imaging results, cytotoxicity and long-term toxicity have not been studied.

4.1 FL and MRI

To meet the requirements of noninvasive, real-time, deep-seated tissue responsiveness for cell tracking technology [120], Song et al. [121] prepared Janus Fe₃O₄@PFODBT-COOH (S _d was 51 nm) from poly[2,7-(9,9-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole](PFODBT, M _w = 10,000–50,000), which exhibited fluorescence property at 680 nm, poly(styrene-co-maleic anhydride) (PSMA, M _w = 1,700) which modified the nanoparticle surface with carboxyl groups to improve stability and prepared Fe₃O₄ nanoparticles by bath sonication. The r ₂ signal intensity thereof seven times that commercial Feraheme at the same Fe concentration, at the same Fe concentration, at the same time, the cells were labelled and tracked by the optical properties of semiconductor material PFODPT, which was higher with a signal-to-background ratio of 2.03 at 250 labeled cells, what’s more, the FL and MRI signals only decreased by about 20% after 10 days. Although the Janus Fe₃O₄@PFODBT-COOH used in the present work achieved the combination of optical and magnetic imaging, it cannot meet the development needs of targeted, multi-mode imaging when it was applied only to the applied imaging after cell labeling.

To improve precise diagnostic and effective therapeutic action, Wang et al. [122] reported a simple method to direct assembly of curcumin (anticancer drug), Ce6 (photodynamic agent), and pH-sensitive molecules into polyphosphazene, and coated on Fe₃O₄ NPs to attain the final Janus NPs (FHCPCe NPs, the average size was 100 ± 10 nm). The M _s and r ₂ values of the FHCPCe NPs were 64.06 emu g⁻¹ and 184.92 mM⁻¹ s⁻¹, respectively, which was higher than commercial Fericia. The anti-cancer results showed a hyperintense area for T ₂-weighted MRI in vivo. More interestingly, over time, the signal at the tumor gradually increased, reaching its maximum value at 8 h, which indicated FHCPCe NPs accumulated at tumor cells because of the targeting properties of polyphosphazene. More importantly, because of Ce6 coupling to the surface of polyphosphatide, easy aggregation of Ce6 in water could be improved, and the fluorescence intensity and the yield of singlet oxygen were significantly increased. Therefore, the FL effect better complements the deficiency of MRI imaging, and a large number of singlet oxygen molecules can kill cancer cells efficiently, thus truly achieving a highly efficient integration of diagnosis and treatment.

4.2 PA and MRI

Janus imaging can facilitate the fast, effective and efficient imaging date, photoacoustic imaging (PA) was favored by researchers as its compatibility [123]. Bell and colleagues [124] developed a novel Janus model (attachment of Flammas774 to Fe₃O₄, with a size of 150 nm, FeO-774) for both PA and MRI. The reason for using Fe₃O₄-774 is that MRI offers complex anatomic details and PA provides biological- and metabolic-related information [125]. The optoacoustic absorption signal for FeO-774 (with a maximum absorption wavelength of 780 nm) showed the intensity of linear curve between concentration and absorption, which proved that FeO-774 was effective for use in suitable PA probes. The r ₂ was 212 mM⁻¹ s⁻¹ for FeO-774, which was higher than commercial Resovist (r ₂ = 151 mM⁻¹ s⁻¹) [126]. Although the results of in vivo imaging indicated that FeO-774 provided not only high-quality PA imaging but also excellent T ₂ imaging, the results proved that FeO-774 was not aggregated in vivo; however, the author did not consider its toxicity.

To compensate for the deficiency of pure T ₂ magnetic imaging, Ren et al. composed core/shell structure Fe₃O₄/Au NPs (of cubic structure with S _d of 51.4 ± 2.8 nm) for dual-modulus imaging of MRI and PA for accurate diagnosis [127]. The magnetic saturation and r ₂ values were respectively 97.8 emu g⁻¹ and 625.1 mM⁻¹ s⁻¹. In vivo for MRI, the tumor area presented a darker magnetic field of vision after injection, which exhibited higher property for MRI. At the same time, PA imaging showed obvious neovascularization in the tumor area. The combination of the two methods shows a certain potential for clinical application.

4.3 CT and MRI

The combination of positron emission tomography (CT) and MRI is more popular in Janus model because MRI can provide images with excellent soft-tissue details [128], whereas CT imaging can provide a high-3D-resolution information of bone and calcification, so as to repair the defects of MRI, and it has a significant effect on early diagnosis of cancer [129].

To overcome the colloidal instability and poor biocompatibility with an organic layer of oleylamine or oleic acid coated Fe₃O₄ nanoparticles [104], Cui et al. [130] used an inorganic aluminum hydroxide layer to coat on the surface of Fe₃O₄ nanoparticles, and studied the influence of the thickness of aluminum hydroxide on MRI imaging. At the same time, bisphosphonate polyethyleneglycol (BP-PEG) was modified with the help of the phosphate radical on BP-PEG and aluminum to form the covalent coordination of hydroxide on fluoride, which can be used to realize CT and MRI imaging together and improve the accuracy of diagnosis. Interestingly, after filtering, the r ₂ signal value of the sample can be increased by at least 1.5 times, which is similar to the results of José Rivas et al. [102]. When the ratio of ferric oxide to aluminum hydroxide is 1:1, the particle size can reach a minimum (21 nm); fluoride can also be effectively adsorbed, which leads to fluoride being located in the liver and spleen rather than being absorbed by bone because of the smaller particle size: this can prolong the blood circulation time [131], so that the sample will not decay in the body for at least 2 h.

To improve the accuracy of diagnosis, Shen and Shi [132] prepared superparamagnetic manganese ferrite (5 nm) using an environment-friendly solvothermal method, and then modified with PEG-dopamine and target material RGD to improve targeting and avoid phagocytosis of RES [133]. With the aid of dopamine and the RGD sulfhydryl group, a ⁶⁴Cu radiolabel was adsorbed and the final product was obtained, which are targeted nanoparticles with CT and MRI effects (⁶⁴Cu-NPs-dopa-PEG-DOTA/RGD, S _d = 26.4 ± 7.5 nm). These 5 nm nanoparticles exhibited high M _s (41.7 emu/g) at 15,000 Oe, and the r ₂ was then 267.5 mM⁻¹ s⁻¹ (v. [Mn + Fe]), which was more than two times higher than Feridex (c.120 mM⁻¹ s⁻¹) [129]. In vivo imaging shows that nanoparticles can effectively gather in tumor areas, and radioactive substances are also more highly concentrated in organs than elsewhere; therefore, CT and MRI imaging effects were significantly improved, which indirectly proves that RGD can indirectly improve CT and MRI effects. In addition, because of the presence of RGD and dopamine, the blood circulation time was significantly increased.

Although MRI/CT dual imaging is realized above, MRI involves single signal imaging, which cannot achieve the accuracy needed for tissue recognition. Zhang et al. [134] wrapped GdF₃ and Fe₃O₄ nanoparticles with PEG to form weighted T ₁/T ₂ signal and CT imaging capabilities to meet the high demand imposed by cancer diagnosis. In the mixed solution of ethylene glycol and water, PEG, Gd³⁺, Fe³⁺, and NH₄F (as a catalyst) were added in turn to form PEG-coated GdF₃:Fe nanoparticles at 100°C (length = 51.9 ± 6.1 nm, width = 31.3 ± 3.5 nm). The M _s of the PEG-GdF₃:Fe NPs (2.38 emu g⁻¹ v. [Gd + Fe] at 20 kOe) was higher than that of KGdF₄ NCs (1.97 emu g⁻¹) [50]. The r ₁ and r ₂ relaxivity values of the PEG-GdF₃:Fe NPs were 3.3 and 36.0 mM⁻¹ s⁻¹, respectively, and the r ₂/r ₁ ratio was 10.2, but PEG-GdF₃:Fe NPs still be used as dual modulus imaging CA, which was analogy with FeCo NPs (r ₂/r ₁ = 9.2) [135]. The blood circulation time increased because of modified PEG [136], and the nanoparticles could accumulate effectively at tumor cells. After 1.5 h post-injection, dual-mode imaging could still be obtained clearly. At the same time, CT revealed that the imaging effect was still better than the clinical iobitridol at low dosage. The above results demonstrated the potential of multi-mode Janus imaging.

4.4 CT, FL, and MRI

Recently, the integration of targeted tumor diagnosis and treatment is a method that combines targeted diagnosis and treatment, which has become a powerful method to improve the cure rate. Janus nanoparticles joining three imaging mediums, CT, MRI and FL, can meet the requirements and show the great potential in imaging, diagnosis, and treatment [137].

Marco and colleagues had recently reported a new Janus NPs owning Au NPs with two enzymatic effectors and mesoporous silica coated β-cyclodextrin, which formed pH-responsive supramolecular nanovalve [138]. Dox loaded on porous structure based on the smart nanovalve successfully evaluated the anticancer effect with HeLa cancer cells in vitro, showing a significantly improved antitumor effect. However, it cannot achieve the purpose of imaging and real-time tracking simultaneously.

Then they reported again [139] on a novel Janus NPs by combining an Fe₃O₄ NPs/mesoporous silica@Au NPs to form core/shell structure, and modified the targeting peptide (cRGD) and a fluorescent dye. The preparation process is as follows: oleic-capped magnetic ferric oxide nanoparticles (Mag 320) were synthesized according to Liong’s method [140], involving cetyltrimethylammonium bromide in a reaction with oleic-capped magnetic ferric oxide nanoparticles in ultrapure water at 60°C under vigorous stirring. After adjusting the pH with sodium hydroxide, TEOS was added, and the magnetic ferric oxide coated with mesoporous SiO₂ (Mag 320@MS) was obtained by electrostatic attraction. Mag 320@MS was emulsified by adding paraffin; after centrifugation and dilution with methanol, Mag 320@MS was reacted with (3-mercaptopropyl)trimethoxysilane, and the product was stirred and compounded with the prepared Au nanoparticles [141] to obtain Janus Au-Magnetic@MS NPs (Janus Au-Mag 320@MS). Catalyzed by NHS and EDAC, Alexa Fluor 647 Hydrazide was modified to Janus Au-Mag 320@MS, then cRGD was modified on the surface in a phosphate buffer by covalently linking, and finally, the fluorescent material was modified on the Janus NPs (RAM, the overall size was 163 ± 2.8 nm). The saturation magnetization value of RAM decreased to 7 emu/g (the M _s of oleic-capped Fe₃O₄ NPs was 33.7 emu/g). Effectively, the low r ₂ and r ₁ values were 13 and 0.3 mM⁻¹ s⁻¹, respectively, and final r ₂/r ₁ ratio was 39, which was deemed acceptable against other reported MNPs [142]. This was mainly because of the ideal spatial structure of mesoporous silica, which could make protons pass through smoothly, thus producing resonance signal; however, because of the influence of more coating layer and other metals, the resonance signal was relatively weakened [141]. The fluorescence emission of RAM was not completely quenched, and the intensity of the fluorescence increased with the increase in concentration, which showed excellent optical tracking. The HU experimental values of RAM also increased with the concentration (linear relationship R ² = 0.9963). No toxicity was found in HEK293, HepG2, and RAW 264.7 at 350 µg/mL of RAM in vitro. After 2 h of intravenous injection, RAM aggregation could be seen on MRI, which was mainly attributed to the effective targeting of rRGD, and the same results could still be seen on PET-CT (the PET-CT signal was more than twice as strong in terms of HU) and fluorescence tracing (no obvious weakness of fluorescence signal was observed). These results demonstrated the potential of RAM in multi-model imaging for diagnosis.

Whether using single modulus, dual modulus, or Janus-multi-modulus, it is necessary to apply ferric oxide to the accurate diagnosis of diseases. The relevant parts of the article are summarized in Table 1. From Table 1, we conclude that from the T ₂ signal to T ₁ signal and then to dual modulus mode, the size of nanomaterials is continuously decreasing, and M _s is correspondingly reduced, which relatively reduced the r ₂ signal and increased the r ₁ signal according to the different imaging methods used. Of course, as the particle size and M _s decrease, this also causes Fe₃O₄ NPs to weaken or even lose some magnetic properties, such as magnetic targeting and magnetic thermotherapy. At present, it is impossible to maintain the excellent magnetic properties on the basis of the dual modulus mode of operation, which needs further research.