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Novel monodisperse FePt nanocomposites for T2-weighted magnetic resonance imaging: biomedical theranostics applications

Given the high incidence and mortality of cancer, current research is focused on designing efficient diagnostic methods. At present, clinical diagnoses are made based on X-ray, computed tomography, magnetic resonance imaging (MRI), ultrasound, and fiber optic endoscopy. MRI is a powerful diagnostic tool because it is non-invasive, has a high spatial resolution, uses non-ionizing radiation, and has good soft-tissue contrast. However, the long relaxation time of water protons may result in the inability to distinguish different tissues. To overcome this drawback of MRI, magnetic resonance contrast agents can enhance the contrast, improve the sensitivity of MRI-based diagnoses, increase the success rate of surgery, and reduce tumor recurrence. This review focuses on using iron-platinum (FePt) nanoparticles (NPs) in T2-weighted MRI to detect tumor location based on dark-field changes. In addition, existing methods for optimizing and improving FePt NPs are reviewed, and the MRI applications of FePt NPs are discussed. FePT NPs are expected to strengthen MRI resolution, thereby helping to inhibit tumor development.


Introduction
Magnetic resonance imaging (MRI) is commonly used for cancer diagnosis, and contrast agents (CAs) play a vital role in MRI applications. [1][2][3] For MRI analysis, the hydrogen nuclei must return to the equilibrium state from the excited state and align with the main magnetic eld. This phenomenon causes two independent relaxation processes with different time constants: T1 and T2 relaxation. T1 relaxation depends on the alignment of the hydrogen nucleus with the main magnetic eld. In contrast, T2 relaxation is related to the disappearance of the phase coherence of the hydrogen nucleus when the external magnetic eld is turned off. MRI analysis methods based on these two relaxation processes have their own suitable CAs. Many T1-or T2weighted MRI CAs based on Gd 3+ , Mn 2+ , and iron nanoparticles (NPs) such as iron oxide (Fe 3 O 4 ) NPs have been developed to improve imaging contrast. [4][5][6] Most clinically used MRI CAs work by shortening the T1 and/or T2 relaxation time of protons inside tissues, causing the signal strength of the T1-weighted image to increase or the signal strength of the T2-weighted image to decrease. Several T1-weighted CAs based on Gd 3+ ion complexes have been widely used in clinical practice. 7 However, low-dose compounds/drugs usually have short circulation times in the body and produce a certain degree of toxicity. These drugs may cause side effects such as nephrogenic systemic brosis and brain deposits. Therefore, researchers have sought other MRI CAs based on candidate materials such as superparamagnetic NPs, particularly iron-based NPs. 8 In the past few decades, some iron-based T2-weighted CAs have been used in clinical trials or been approved by the U.S. Food and Drug Administration (FDA). 9 Fe 3 O 4 in iron-based NPs is already used as a commercial T2weighted MRI contrast agent. 10 However, its main drawbacks are the agglomeration of NPs, wide particle size distribution, and poor zeta potential value. 11 Fe 3 O 4 NPs also lack good colloidal stability, and the repulsive force is insufficient to prevent aggregation. 12 This problem can be attributed to the problems caused by the reaction time and temperature of Fe 3 O 4 NPs during the coprecipitation process. Controlling the synthesis procedure is necessary to stabilize Fe 3 O 4 NPs and reduce their size by modifying their surface with biocompatible materials.
In contrast, FePt has better colloidal stability. Its uniform size distribution is more suitable as a T2-weighted MRI contrast agent. In the presence of oleic acid and oleylamine stabilizers, monodisperse FePt NPs are synthesized by reducing platinum acetylacetonate and the decomposition of iron pentacarbonyl.
The composition of FePt NPs is easy to control, the size can be adjusted in the range of 3 to 10 nm in diameter, and the standard deviation is less than 5%. 13 However, due to the inherent dark signal of NPs and MRI sensitivity artifacts, it is difficult to distinguish small, early-stage tumors from hypolipidemic areas. 14-16 Therefore, the clinical applications of these iron-based NPs remain limited. This problem has prompted scientists to consider accumulating more MRI CAs in specic target tissues (e.g., tumor tissues). [17][18][19] For instance, to develop MRI CAs that can target specic tissues, NPs have been modied with specic antibodies that allow the NPs to easily penetrate the centers of lesions to improve the MRI image contrast. [20][21][22] Magnetic NPs (MNPs) can enhance image contrast and carry cargos such as chemotherapeutic drugs to monitor the development of diseases during treatment and make treatment decisions in real-time. [23][24][25]

FePt NPs as MRI CAs
MNPs have attracted considerable attention because of their magnetic susceptibility, biocompatibility, stability, and other advantages. MNPs mainly include metal oxide NPs (e.g., Fe 3 O 4 ), 26 metal NPs (e.g., Fe and Co), 27 and metal alloys (e.g., FePt and FeCo). 28 Fe 3 O 4 NPs have many advantages as MRI CAs, including strong superparamagnetism, good biocompatibility, easy biodegradability, and simple synthesis. Therefore, Fe 3 O 4 NPs have been used in MRI-based diagnosis and treatment. [29][30][31] However, when used alone, Fe 3 O 4 NPs are quickly cleared by the liver and spleen due to their size. 32 Metal alloy NPs with better magnetic properties, such as FePt NPs, have received increasing attention from many researchers. FePt NPs can exhibit excellent superparamagnetism and have chemical stability against oxidation. In addition, FePt nanoparticles with adjustable size and shape can be prepared and modied for different biomedical applications. [33][34][35] FePt NPs have three different lattice structures: disordered face-centered cubic, ordered facecentered tetragonal and L10 phase. Existing methods for synthesizing FePt nanostructures include physical techniques such as mechanical deformation, arc melting, vacuum evaporation (sputtering and thermal evaporation), laser ablation, chemical methods, physical/chemical methods and electrodeposition. FePt NPs are more stable against oxidation than FeCo NPs and have the advantage of maintaining long-term stability. 36 The sizes and shapes of FePt NPs can be adjusted for various uses in biomedicine. [37][38][39] The modication of FePt NPs can help improve the T2 image contrast and effectively reduce the side effects, resulting in better MRI CA candidates. This review article compiles the research progress made related to FePt NPs and their application as MRI CAs.

Toxicity assessment of FePt NPs
Compared with other inorganic nanomaterials, the key to applying iron-based NPs such as Fe 3 O 4 NPs and FePt NPs in essential clinical research lies in the natural integration of such NPs into physiological tissues. 40 Through human tissue proteins such as ferritin, transferrin, and hemosiderin, iron and its oxides can be metabolized, stored, and transported in the organism. [41][42][43] The magnetic moment of Fe 3 O 4 NPs will uniformly separate under an external magnetic eld but agglomerate without the magnetic force. By comparison, FePt NPs with a particle size of less than 10 nm exhibit a single magnetic domain and have superparamagnetic properties like Fe 3 O 4 NPs, increasing their diversied clinical applications in medicine. The FePt NPs can disperse in a simulated biological environment without an external magnetic eld, which has expanded their diversied clinical applications in medicine. Based on recent research, the main potential concern is the application of NPs in biomedicine. [44][45][46][47] The toxicity of NPs is mainly attributed to the following: (1) the results are confusing and contradictory due to the inconsistent experimental standards; (2) the surface properties and size of the NPs will affect the toxicity by affecting the distribution in organs and metabolic time; (3) cell experiments: the results of animal experiments cannot be extended to humans; (4) the long-term toxicity is difficult to detect and study. The organism itself can relieve the toxicity of NPs, such as using protein crowns and liver and kidney metabolism. 48 On the whole, the toxicity of NPs is relatively low compared to organic solvents, including carcinogenicity, immunotoxicity, and genotoxicity. 49 Aer the organism degrades FePt NPs, it is mainly metabolized by separating iron ions from platinum. The FDA has approved the low toxicity of iron ions for use in humans for decades, so the main concern is the toxicity of platinum. Platinum itself is very similar to gold, with good biocompatibility, no biological toxicity, and chemical stability. Iron is a metal with excellent magnetic properties, so FePt nano-alloy particles combine the advantages of both. Nanomedicine research has as far as possible through various cell and biological experiments proved that the biocompatibility is good. However, when performing experiments, only a tiny amount of NPs is injected. The selected materials do not contain highly toxic elements, so it is easy to obtain non-toxic results through experiments. In many cases, the dispersibility of NPs directly affects the data results, and vascular embolism caused by aggregated NPs is the leading cause of death in experimental animals. This phenomenon also shows that more research is needed to conrm that these nanomaterials are nontoxic and can be used in clinical medicine. Thus, the carcinogenicity, immunotoxicity, and genotoxicity of NPs require more experiments in the future to give answers to claim they are "safe."

Application of FePt NPs as MRI CAs in biomedical diagnosis and treatment
FePt NPs have high crystallinity, suitable magnetic properties, and uniform sizes, which help avoid their rapid removal from the body and thus improve MRI contrast. Modifying the FePt NP surface can effectively improve the biocompatibility, dispersion, and stability of the NPs for biological applications. For instance, adding gold (Au), silicon dioxide (SiO 2 ), and graphite shells can increase biostability. The addition of degradable biological polymers, including dextran, polyester acid, proteins, and amphiphilic polymers such as polyethylene glycol (PEG), can facilitate the quick dispersion of FePt NPs in the simulated biological environment (Table 1).

Polymer-modied FePt NPs with improved particle dispersion
Slabu et al. mixed hydrophobic FePt NPs with functionalized polymaleic anhydride polymers and dried them to construct hydrophilic FePt NPs that could be redispersed in an aqueous solution.
The preparation process is shown in Fig. 1a. 50 Dynamic light scattering (DLS) analysis showed that the sizes of the FePt NPs coated with amphiphilic polymers were between 7.2 and 14.2 nm regardless of whether the NPs were dispersed in water or PBS, demonstrating the stability of the FePt NPs. Compared to other superparamagnetic iron-based NPs of the same size, the saturation nanoprobe using a one-pot synthetic method. They modied it with glutathione (GSH), effectively improving the particle dispersion and biocompatibility (Fig. 1c-e). 51 According to the experimental MRI results shown in Fig. 1f 52 The preparation process is shown in Fig. 2a. According to 3T MRI experiments, the  synergistic effect of Mn and Fe greatly enhanced the T2 image contrast, with r 2 reaching 8.14 mM À1 s À1 . The released Mn 2+ ions also enhanced the T1-weighted contrast, as shown in Fig. 2b and c. FePt@MnO will release iron and manganese ions into the cell environment aer endocytosis. These ions will then interact with antioxidant enzymes such as GSH in the cell, leading to an imbalance in the production and degradation of reactive oxygen species (ROS). This imbalance will trigger the iron-mediated apoptosis reaction (ferroptosis), causing cell death. As shown in Fig. 2d, aer intravenous/intratumor injection, the uorescence was signicantly higher in the FMDF NP group than the control group, indicating a sharp increase in the ROS level in the tumor tissue than in the normal tissue. In vivo experiments using mouse mammalian gland tumor cells, 4T1-bearing Balb/c mice, revealed that the tumors were inhibited in the FMDF NP group, as shown in Fig. 2e and f. Based on a biodistribution experiment, the position of the cancer was different. The results also proved that the targeting effect of FMDF could make the NPs accumulate in the tumor tissue. The aggregation of multi-iron and manganese alloy conrmed folic acid (FA) ability-modied FePt@MnO to target tumors. Kaolinite, a hydrophilic material with many hydroxyl groups on its surface, is widely used to alter iron NPs. Chan et al. modied FePt NPs with kaolinite to form a sandwich-like FePt@Kao nanostructure. The bulk kaolinite in FePt@Kao is split into a layered structure by the interface-active agent cetyltrimethylammonium bromide. 53 Aer ultrasonic and heat treatment, the kaolinite layer gradually peels off into a uniform nanomaterial. Kaolinite has a stable chemical structure, strong adsorption capacity, high biocompatibility, and low toxicity. The layered kaolinite structure can be loaded with many different substances, including chemotherapeutic drugs, doxorubicin (DOX), and FePt NPs. Furthermore, the drug release rate can be controlled through this structure (sandwichlike structure of FePt embedded in kaolinite), as shown in Fig. 2g. FePt NPs containing DOX was loaded into the layered space of 20 mg kaolinite to make it highly concentrated in composite NPs. The FePt NPs are subjected to uniaxial compression perpendicular to the montmorillonite layer, and the mechanical stress causes the FePt NPs to form magnetic dipoles. This phenomenon causes the NPs to rearrange so that they are parallel to the direction of compressive stress. Therefore, the saturation magnetization is signicantly increased in the magnetization direction, and the MRI signal is also enhanced (r 2 ¼ 29.32 mM À1 $s À1 ; Fig. 2h-j).

A composite composed of two iron nanomaterials for obtaining stable MRI images
Strong magnetization is one of the necessary conditions for obtaining strong MRI contrast. Although metallic NPs have high magnetization energy, they are easily oxidized in biological environments, resulting in instability. Gao et al. found that coating metal NPs with a layer of polycrystalline Fe 2 O 3 or Fe 3 O 4 can signicantly improve their stability. 73 Liu et al. developed FePt@Fe 2 O 3 core-shell magnetic NPs with FePt NPs as the core and Fe 2 O 3 as the shell; PEG-FA was added to reduce cytotoxicity and non-specic binding. 46 The addition of FA allows the effective targeting of folate receptor-positive cancer cells such as papilloma KB cells (Fig. 3a-c). Based on DLS analysis, the FePt@Fe 2 O 3 -PEG NPs have a size of approximately 51 nm. Fig. 3d and e show that as the iron concentration increased, the T2weighted MRI image became darker (T2 relaxation rate ¼ 91.9 mM À1 s À1 ). The intravenous injection of the FePt@Fe 2 O 3 -PEG NPs into Balb/c mice signicantly reduced the T2-weighted signal intensity of the tumor. In vivo experiments demonstrated that the FePt@Fe 2 O 3 -PEG NPs provide obvious tumor contrast (Fig. 3f). According to the results of cell viability and mouse experiments, when applied to treat KB cells, high concentrations of the FePt@Fe 2 O 3 -PEG NPs did not produce any apparent cytotoxicity or cell membrane damage (Fig. 3g and h). Yang et al. used PEG-FePt@Fe 3 O 4 MNPs to treat mouse breast cancer. MNPs with PEG chains can increase the internalization ratio of NPs, prompt the therapeutic effect, improve the image contrast of tumors, and induce tumor cell death through local temperature increases. 57 The application of PEG-MNPs signicantly reduced the tumor volume; aer repeated hyperthermia for two weeks, the tumor was approximately 4.5 times smaller than the control. Yang et al. successfully applied FePt@Fe 3 O 4 NPs with hyperthermia to slow tumor growth for cervical cancer treatment, as shown in Fig. 3i.
The therapeutic effect is primarily attributed to the high magnetic heat transfer capacity of the PEG-MNPs, which causes thermal ablation and enhances the anti-tumor effect. 56 Kim et al. found that 3,4-dihydroxyphenylacetic acid (DOPAC) was a better ligand than dimercaptosuccinic acid (DMSA) for functionalizing FePt@Fe 3 O 4 core-shell NPs. 58 Compared to DMSA, DOPAC undergoes fewer oxidation reactions, increasing the magnetization energy (Fig. 3j). As shown in Fig. 3k, DOPAC-FePt@Fe 3 O 4 results in T2 contrast enhancement with an r 2 of 131.5 mM À1 s À1 , suggesting that DOPAC-FePt@Fe 3 O 4 is a potential tool for magnetic hyperthermia.

Composite FePt NPs for enhancing the therapeutic effect
Under an external magnetic eld, free electrons can be excited on the surfaces of FePt NPs. This process releases thermal energy via magnetothermal conversion, which causes cancer cells to enter the apoptotic process. Thus, FePt NPs can not only be used for T2-weighted MRI diagnosis, but they can also be used simultaneously for treatment based on the production of magnetic heat, which is called magnetic uid hyperthermia (MFH). 74 Kostevšek (2018) et al. coated multinuclear FePt NPs with SiO 2 followed by a two-step inoculation with Au, thereby enhancing the heat generation of the nanosystem. 59 Coating the FePt NPs with SiO 2 can improve the biocompatibility of the NPs. Lai et al., who previously coated FePt NPs with silica to enhance biocompatibility, also used this synthetic method.
The transmission electron microscopy images of FePt NPs shown in Fig. 4a-c show that the FePt cores were coated with SiO 2 to form FePt/SiO 2 NPs with an average diameter of 40 AE 3 nm. Au seed crystals of approximately 1-3 nm were formed on the SiO 2 surface during the rst Au seeding step. Aer the second Au seeding step, Au particles of 6-10 nm size were found on the SiO 2 surface. The longitudinal (T1) and transverse (T2) relaxation rates of the SiO 2 -coated multicore FePt NPs were 1.5 times lower than those of the FePt NPs without SiO 2 . However, when the FePt/SiO 2 /Au NPs were modied with PEG, the coating thickness increased, and the r 2 value decreased by approximately 1.85 times. Due to the signicant distance between the water protons and the superparamagnetic core, the magnetic eld decreased as the distance from the core material increased. In turn, the relaxation time decreased, affecting the r 1 and r 2 values, as demonstrated by in vitro MRI experiments (Fig. 4d). In addition, modifying the surfaces of the FePt NPs with Au could effectively improve the photothermal reaction. As shown in Fig. 4e, as the concentration of FePt/SiO 2 /Au NPs increased, the heating rate increased linearly, demonstrating the potential of the FePt/SiO 2 /Au NPs as photothermal agents. According to the in vitro cell model analysis, poorly differentiated cancer cells underwent more vital endocytosis than well-differentiated normal urothelial cells. The results indicate that transitionalcell papilloma RT4 cells can internalize more NPs than normal porcine urothelial (NPU) cells ( Fig. 4f and g). Thus, the hybrid NPs show high selectivity for cancer cells, demonstrating their excellent potential for cancer diagnosis and the development of anti-cancer therapies in the future.

Modication with active targeting molecules enhances the accumulation of FePt NPs in tumors
The use of amphiphiles to modify the surfaces of FePt NPs is one of the most effective and widely used methods to improve the dispersibility of MNPs. Modication with amphiphiles can ameliorate the reduced magnetic effect and cytotoxicity caused by the aggregation of NPs with only hydrophilic coatings. Yue et al. loaded PEG and FA on graphene oxide (GO), with abundant carboxyl and hydroxyl groups on the surface, making it stable in an aqueous solution. The researchers then loaded FePt-DMSA on the GO nanosheets to form FePt-DMSA/GO-PEG-FA NPs. 69 The T2-weighted image shows an inverse concentration-dependent MRI contrast, and the r 2 value is 12.425 mM À1 s À1 , suggesting that the FePt/GO composite nanoassemblies are good MRI CA candidates.
Moreover, since FA receptors are highly expressed in malignant tumor cells, the FePt-DMSA/GO-PEG-FA NPs strongly accumulate in breast cancer MCF-7 cells (Fig. 5a-c). At the low pH of the tumor environment, iron ions are released and catalyze ROS formation, inducing cell death. To conrm the therapeutic effect of the NPs, mimicked human 4T1 breast cancer cells were planted under the skin of mice. The tumors were signicantly inhibited by treatment with the FePt/GO composite nanoassemblies, as shown in Fig. 5d. The difference in the tumor size aer nanocomposite treatment can be seen in the T2 MRI image (Fig. 5e).

Modication of FePt NPs with organic molecules to enhance biocompatibility
Although coating the surfaces of FePt NPs with iron oxide and other inorganic materials can effectively improve the MRI image contrast, doubts remain about whether NPs with exogenous coatings can be delivered to normal tissues and organs. Moreover, iron oxide has the disadvantage of being easily corroded. Therefore, many researchers have coated FePt NPs with biological molecules that do not have any safety concerns. Chou et al. modied the FePt NP surface with cysteamine (Cys) to improve cell endocytosis. 70 The exposed amine groups are connected to anti-Her2 antibodies, allowing the targeting of cancer cell lines with high Her2/neu expression, as shown in Fig. 6a. The experimental results in Fig. 6b and c show that the 12 nm FePt-anti-Her2 NPs have high magnetic susceptibility and signicant MRI contrast. Other researchers have also applied Cys as a biosafety modication. Sun et al. prepared FePt-Cys NPs with a size of approximately 254 nm. The T2-weighted images (Fig. 6d and e) show iron concentration-dependent image contrast. Although the r 2 value of the FePt-Cys NPs is not as high as FePt NPs only, the coating of iron with other molecules can avoid the surface of FePt-NPs to oxidize. Still, the use of these NPs provides good biocompatibility and is less likely to damage the part applied to the brain tissue. 45

Conclusion & future perspectives
We have reviewed progress in the modication of FePt NPs over the past few years. FePt NPs are attractive nanoplatforms with high magnetization values and higher stability than other metal NPs. 66,75 Many studies have demonstrated that FePt NPs can enhance the contrast of MRI images. However, surface coatings are needed to improve particle dispersion and reduce toxicity under physiological conditions. Efficient surface coatings can also increase the T2 contrast of FePt NPs to facilitate diagnostic analysis.
Encapsulating FePt NPs in amphiphilic polymers can prevent them from aggregating and releasing ferroportin ions in aqueous environments, 76 thus helping in maintaining the magnetization value and increasing the biocompatibility and safety of the FePt NPs. Therefore, one of the most basic methods to modify FePt NPs is to coat them with amphiphilic polymers. Coating FePt NPs with Au, SiO 2 , GO, and biodegradable polymers can also effectively improve the stability of the FePt NPs. Furthermore, the safety of the NPs can be improved by modi-cation with amino acids, allowing the NPs to be used in diagnosis and therapy for conditions such as brain tumors. However, FePt NPs have the disadvantage of being quickly oxidized; modifying the FePt NPs with iron oxide can stabilize their structures, increase the magnetization, and enhance the T2 MRI contrast. Moreover, kaolinite-based FePt NPs with sandwich-like designs can alter the magnetic dipole arrangement of iron and platinum to increase the saturation magnetization and effectively improve the MRI image intensity. 64,65,77 FePt NPs can convert superparamagnetism into ferromagnetism through rapid changes in the magnetic eld, resulting in the loss of magnetic energy and heat energy generation (i.e., the MFH effect). Based on the sensitivity of tumors to heat, exploring whether NPs can serve as local heating agents is an attractive strategy. The modication of FePt NPs with folate or antibodies can allow the NPs to specically target tumors. When the NPs are concentrated at the tumor site, they can also effectively improve the T2 contrast at the tumor location. Chemotherapeutic drugs can also be loaded into FePt NPs to

Conflicts of interest
There are no conicts to declare.