# Applications of Iron Oxide Nanoparticles against Breast Cancer

Authors: Hessam Hosseinkazemi; Saeed Samani; Andrew O’Neill; Mahdieh Soezi; Mohsen Moghoofei; Mohammad H. Azhdari; Farzaneh Aavani; Abolfazl Nazbar; Saeed Heidari Keshel; Mohammad Doroudian
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Breast cancer is the most common cancer diagnosed in women, with an estimated 12% of women in the United States affected during their lifetime. Researchers have demonstrated that early detection, diagnosis, and treatment are pivotal to increasing survival. The advent of nanotechnology has yielded several novel advances and available modern methods within the clinic to detect and treat breast cancer. Inorganic nanoparticles are broadly utilized for cancer diagnosis and therapeutic purposes. Interestingly, these nanoparticles can also be attached to tumor-specific ligands and used to deliver chemotherapeutic or hormonal agents with high levels of tumor selectivity. Iron oxide nanoparticles are one of the most commonly used nanomaterials, which have attracted much attention to detect and treat breast cancers, owing to their superparamagnetic characteristics. Computerized tomography and magnetic resonance imaging (MRI) utilizing iron-based magnetic nanoparticles are promising approaches for the radiological detection of breast cancer. Here, we discuss the roles and recent applications of iron oxide nanoparticles in diagnosing and treating breast cancer.


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## Body

## 1. Introduction

Cancer is the second cause of death worldwide, and it is expected that it will become the most common cause of morbidity in future years [1]. Currently, conventional cancer treatments are limited to surgery, radiotherapy, chemotherapy, and immunotherapy [2]. Unfortunately, these approaches have limitations, including nontargeted delivery of antitumor drugs, insufficient drug concentrations at tumor sites, restricted monitoring of therapeutic responses, and severe associated side effects [3]. Breast cancer is the most commonly diagnosed cancer in women, which develops due to uncontrolled division and growth of cells within the breast [4]. The current paradigm of breast cancer pathogenesis is that invasive cancer occurs as a result of a series of molecular variations at the cellular level, resulting in uncontrolled growth in breast epithelial cells [5]. Breast cancers expand within the breast tissue and may be diagnosed during a screening checkup, either before symptoms have developed or after a lump has been discovered [6].According to the American Cancer Society (ACS) statistical evaluations, there will be an estimated 1.9 million new cancer cases and about 608,000 cancer deaths in 2021 in the U.S. alone [7]. As a result of the profound impact of cancer, numerous investigations have focused on cancer patients to find factors affecting the onset, progression, and development of various cancers (Figure 1). Nanotechnology is the design, characterization, fabrication, and application of nanoscaled engineering, healthcare, drugs, biology, chemistry, material science, and physics. In this interdisciplinary field, controlling the shape and size of material at the range of 1–100 nm strongly influences physical and chemical properties [8]. Some studies have proved that SPIONs higher than 50 nm are rapidly cleared from the bloodstream by the phagocyte system [9, 10]; on the other hand, USPIONs smaller than 50 nm can easily escape and have a higher chance to deliver to the targeted tissue including tumor [11, 12]. Correspondingly, Zhang et al. investigated the impact of USPION properties, including size and surface modification, on nanoparticle uptake between healthy and cancer cells. The results showed that PEGylated USPIONs were endocytosed neither by healthy nor by breast cancer epithelial cells, causing a decrease in MRI contrast. Also, it was indicated that 30 nm nanoparticles had better uptake than the smaller ones in the size of 10 nm [13].Figure 1
Number of published original articles related to cancer and breast cancer based on Scopus database.In medicine, nanotechnology created the “nanomedicine” concept, investigating biological and physiological systems at the nanoscale range. Nanomedicine for cancer treatment presents numerous advantages, including controlled biodistribution of nanosystem for active and passive targeting, avoiding biological barriers to maintain bioavailability and pharmacokinetic properties of drugs, and modification by multiple targeting moieties site-specific targeting [8, 14]. Thus, nanotechnology represents a potential alternative for conventional cancer diagnosis and treatment modalities (Figure 2) [15, 16]. Aiming to speed up the integration of nanotechnology into cancer research, the National Cancer Institute (NCI) of the National Institutes of Health (NIH) founded the NCI Alliance for Nanotechnology in Cancer program in 2005. As a result, many nanotechnologists and biologists working on cancer have gathered together to find suitable treatments for different types of cancers [17].Figure 2
Applications of iron oxide nanoparticles (IONPs) against breast cancer.Nanotools are engineered individual multicomponent particles that can be exploited for diagnostic and therapeutic actions [18, 19]. During recent decades, nanotools have emerged to provide an efficient alternative in breast cancer management. They are exploited for both diagnostic and therapeutic actions, such as drug/gene delivery, laser-assisted therapy (e.g., photodynamic therapy (PDT) and photothermal therapy (PTT)), and imaging modalities (e.g., MRI and positron emission tomography (PET)) [18, 20]. Available nanotools for early cancer detection and targeted therapy can be categorized into organic and inorganic particles. Organic nanotools include liposomes, polymeric micelles, dendrimers, and nanocantilevers [21]. Inorganic nanotools include silica nanoparticles, carbon nanomaterials, nanographene, gold and silver nanoparticles, magnetic nanoparticles, and quantum dots which are widely employed for cancer treatment [22]. In addition, inorganic nanoparticles can be conjugated with specific targeting ligands for selective delivery of drugs and hormones to target sites since they help track and monitor the delivery system into the target site. Due to their properties, nanotools can be employed for PDT, radiotherapy, and radiofrequency treatment and as theranostic agents [23–25]. Iron oxide nanoparticles (IONPs) have been widely utilized to detect and kill cancer cells with an elevated temperature, for instance, hyperthermia of breast cancer tumors. These inorganic particles might attach to cancer cells and get magnetized after exposure to the magnetic field [26]. IONPs can be conjugated with anticancer drugs, antibodies, nucleotides, enzymes, and proteins to deliver them to the specific target site using an external magnetic field [16]. In this review, we provide a bird’s-eye view of the application of iron oxide nanoparticles as magnetic particles to detect, monitor, and treat breast cancer.


## 2. Iron Nanoparticles: A Brief on Synthesis Methods and Types

Iron nanoparticles are synthesized via chemical, physical, and biological processes (Figure3). Chemical methods are popular due to low production cost, high yield and management of particle size, composition, and shape [27–29]. In this procedure, iron oxide nanoparticles are produced through the coprecipitation of Fe2+ and Fe3+ by the addition of a base [30, 31]. This technique includes the sol-gel method, oxidation, chemical coprecipitation, hydrothermal, flow injection, electrochemical, aerosol/vapor phase, sonochemical decomposition, simple fluid method, and nanoreactors. On the other hand, gas deposition and electron beam lithography are examples of the physical process that are not very prevalent because of the poor control of particles’ size [27, 32]. The biological method exploits the bottom-up approach, i.e., assembling atoms and molecules to make nanostructures. Although this process is safe and economical, it is prolonged and lacks a desirable control over the particle size [33].Figure 3
Methods for synthesizing iron oxide nanoparticles, taken from [27].


## 3. Applications of Iron Oxide Nanoparticles

### 3.1. Imaging Modalities

#### 3.1.1. MRI

Early detection of breast cancer, which is crucial to limit progression to the metastatic phase, is routinely achieved using mammography, ultrasound, and MRI [34, 35]. MRI takes advantage of protons’ magnetic properties to make high-resolution images from different body parts when patients are situated in a magnetic field. However, this technique might give false positives, especially at ductal carcinoma in situ (DCIS) diagnosis [36, 37]. Han et al. have developed a method to synthesize magnetic-fluorescent iron oxide-carbon hybrid nanomaterials using a one-pot reaction. These nanoparticles not only have many magnetic features for cancer cell detection but also; thanks to their fluorescent characteristics, colorful imaging of cells is possible as well [38].Moreover, since a cluster of differentiation 44 (CD44) is usually overexpressed in aggressive breast cancer cells [39, 40], the hybrid nanoparticles were conjugated with CD44 monoclonal antibodies to provide fluorescent/MRIs with an enhanced track of cancer cells [38]. MUC1, a compound involved in cell-cell and cell-matrix interaction [41], is another overexpressed glycoprotein in adenocarcinomas such as breast cancer cells [42]. Relying on this fact, Khaniabadi and coworkers have detailed the development of a magnetic contrast agent comprised of superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with C595 monoclonal antibody to target MUC1 overexpressed MCF7 cells. These nanoprobes showed excellent biocompatibility with low cytotoxicity. In addition, magnetic resonance contrast agents, at high concentration, decrease T1 and T2 relaxation times, which enhance MRI efficacy [43].Rezayan et al. reported surface modification of Fe3O4 nanoparticles with polyethylene glycol diacid and dopamine as a linker to increase the biocompatibility and dispersity of the particles. Various concentrations and time points were examined to assess the efficacy of the nanoparticles for MRI application. The results demonstrated a strong reverse relationship between the agents’ concentration and relaxation time, leading to improved magnetic resonance signal. Moreover, MTT assays on MDA-MB-231 cancer cells were performed at various concentrations of modified and unmodified nanoparticles for 24 and 48 hours. The results demonstrated that the PEGylated IONPs were less cytotoxic due to the less Fe ion release from particles and lower chemical activity. The cellular absorption of coated nanoparticles was 80% comparing to the unmodified ones with 9% [44].


#### 3.1.2. Computerized Tomography (CT)

CT applies X-ray to provide two-dimensional images of the body, and different techniques are used to make three-dimensional images to detect the site of the lesion. However, this method lacks a desirable resolution and needs an intravenous injection of a contrast agent to enhance the image quality [45]. To provide high-resolution and high-sensitive images, Deng et al. synthesized a novel combination of ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) with cyclic arginine-glycine-aspartate (cRGD) and radiolabeled to use as bimodal imaging agents for single-photon emission computerized tomography (SPECT) and MRI [46]. Since αVβ3 receptors are overexpressed in cancer and endothelial cells in breast cancer [47], cRGD was used due to its high affinity to these receptors. They were coated with carboxymethyl dextran to modify the nanoparticles, conjugated with cRGD peptides via the carboxyl groups on the surface, radiolabeled with iodine. These radiolabeled nanoparticles had high tumor absorption, moderate liver elimination, and long circulation half-life. However, iodine was not entirely appropriate as a radioisotope component due to low gamma-ray emission [46].


#### 3.1.3. Combined Imaging Approaches

Imaging techniques have been integrated in recent years to provide an optimal balance of sensitivity and specificity, as well as superior quality in cancer diagnosis [48, 49]. Using this combination, many researches have been performed to investigate the application of iron oxide nanoparticles in the detection of breast cancer. For instance, in a study, Thin et al. introduced a novel combination imaging technique of MRI, SPECT, and bioluminescence imaging (BLI) with adipocyte-derived mesenchymal stem cells (ADSCs) [50]. Mesenchymal stem cells have shown great potential for the treatment of numerous diseases [51], particularly as an assistive system to integrate tumors, as they are easily extracted from various sources and can be genetically manipulated [52]. The ADSCs were radiolabeled with indium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid- (DOTA-) SPIONs and injected both intravenously and intracardially into the mice with 4T1 breast tumor. This study shed light on the importance of stem cell therapy and stem cell tracking in vivo.A study by Kanwar et al. evaluated antitumor and imaging characteristics of Fe3O4-saturated bovine lactoferrin nanocapsules (FebLf NCs) in BALB/c nude mouse xenograft model. The results demonstrated that the FebLf NCs were absorbed into the breast cancer cells via the surface receptors (LRP1, LRP2, TFR-1, and TFR-2) and induced cell death. Moreover, they provided near-infrared images demonstrating the efficacy of these particles in tumor growth control [53]. In a similar study, de Souza Albernaz and colleagues produced IONPs coupled with diethylenetriaminepentaacetic acid (DTPA) and trastuzumab. They labeled them with two radioisotopes of Gallium-68 and Technetium-99 to conduct PET and SPECT imaging. In vivo evaluation in 6-week BALB/c nude mice showed that more than 80% of the dual-modality agents were removed in healthy mice via the renal system, while in the xenograft mice, the tumors internalized a considerable amount of 1.98 μCi due to their HER2 overexpression [54].To track anticancer drugs in a shorter time and a noninvasive manner, Wang et al. reported synthesizing fluorine-18-labeled arginine–glycine–aspartic acid- (RGD-) coupled ultrasmall iron oxide nanoparticle (18F-RGD@USPIO) using click chemistry. They utilized PET/CT and MRI techniques to monitor the antiangiogenic influence of intraperitoneally injected bevacizumab anticancer drugs over seven days. In this study, in vitro testing was conducted on MDA-MB-231 cell lines, and in vivo experiments were conducted using MDA-MB-231 xenograft-bearing mouse models. The results proved high stability, low cytotoxicity, and excellent biodistribution of the agents. Moreover, PET/CT and MRI imaging confirmed a lower 18F-RGD@USPIO coupling after bevacizumab injection due to the tumor’s low vascular density [55].


### 3.2. Magnetic Hyperthermia

Magnetic hyperthermia is a therapeutic process by which the internal temperature of cancer-affected tissues is raised to 43–46°C to kill cancer cells directly [56]. It is administered together with other cancer treatment modalities to control tumor growth [57]. The application of iron nanoparticles can avoid damaging the surrounding tissue since they help increase the temperature in the nanoscale within the targeted tissue [56]. Magnetic nanoparticles can generate heat inside a tumor when placed in an alternating magnetic field (AMF) via Neel and Brownian mechanisms [58]. Hyperthermia executes cancer cells via various paths such as necrosis, apoptosis, protein denaturation, and immune system reaction to heat shock proteins produced due to the stress on cancer cells [56, 59, 60]. Recently the application of iron oxide nanoparticles for hyperthermia has received much attention [61–64]. Munir et al. reported the coating of IONPs with malic and citric acid, which resulted in increased magnetic polarization and dimension reduction. After injecting the modified nanoparticles into the breast tumor in the mice, the animal models were exposed to a 15 mT magnetic field and frequency of 100 kHz for one hour. The histological analysis indicated that the AMF caused apoptosis of the cancer cells in the tumor [65]. Cędrowska et al. reported a novel conjugation of trastuzumab monoclonal antibody (Herceptin commercial drug) on SPIONs using 3-phosphonopropionic acid (CEPA) and Actinium-225 isotope as an emitter. The 225Ac@Fe3O4-CEPA-trastuzumab nanoparticles can specifically link to HER2 receptors which are overexpressed on the surface of the breast and ovarian cancer cells. It has been observed that these nanoparticles could target cancer cells and represented radiotherapy and hyperthermia functions [66]. Zuvin et al. developed poly (acrylic acid)-coated USPIONs, labeled with anti-HER2 antibodies to target breast cancer cells, and exposed them to a magnetic strength as low as 0.8 kAm-1 preventing eddy current, which can have detrimental effects on normal tissues. Following in vitro evaluation, it was demonstrated that although the synthesized nanoparticles were not cytotoxic, a considerable reduction in cell proliferation and cell death was spotted among MDA-MB-453 cancer cells after exposure to the magnetic field [67]. In a similar study by Almaki et al., core-shell magnetic nanoparticles coated with polyethylene glycol (PEG) with an average size of less than 150 nm were produced, and trastuzumab was immobilized on the surface of the nanoparticles. To investigate hyperthermia treatment in vitro, MTT assays were performed on different cell lines showing nontoxicity. The results revealed nanoparticle accumulation within the tumor at levels four times higher than the level of accumulation within normal tissues, confirming the considerable potential of the magnetic tumor-targeting hyperthermia concept for breast cancer treatment [68]. A variety of mammalian cells, including cancer cells, express CD-44 antigen, a glycoprotein responsible for different cell behavior, such as proliferation, differentiation, and cell adhesion [69]. In a recent report, hyaluronic acid that is a ligand for CD-44 was covalently immobilized on graphene oxide particles; moreover, doxorubicin and paclitaxel as chemo drugs were added separately to this combination to provide drug-targeting agents. Additionally, to provide hyperthermia functionality, iron oxide nanoparticles were inserted and composited with them. The cytotoxicity assay on MDA-MB-231 and BT-474 breast cancer cell lines proved the high cytotoxic effect on MDA-MB-231 cells with CD44 expression, but not on BT-474 cells having not CD44 [70]. In another interesting study, multifunctional Off-On nanoparticles were synthesized using IONPs as nanocores and poly (lactic-co-glycolic acid) (PLGA) as the nanoshell. In this study, an anticancer drug was packed between core and shell, and polyvinylpyrrolidone (PVP) was added to the core-shell particles. This pH-responsive structure is in the Off mode when circulating inside the body and in the On mode when they enter the cells and places them in an acidic condition causing drug releases. The hyperthermia contributed to suppressing cell growth and increasing the temperature from 37 to 42°C leading to a faster PLGA degradation causing more tamoxifen release and MCF7 apoptosis [71].In a combinational therapy, iron oxide nanoparticles were coated with gold (Au@IONPs) and exposed to radiofrequency (RF) waves and X-ray radiation to provide both RF hyperthermia and radiotherapy for MCF7 apoptosis. The results showed a significantly improved radiotherapy effect when accompanied by RF hyperthermia [72]. In an experiment conducted by Salimi et al., iron oxide nanoparticles were used for magnetic hyperthermia treatment of breast cancer in BALB/c mice. In this study, the IONPs were functionalized with the fourth generation (G4) of poly amidoamine (PAMAM) dendrimers (G4@IONPs) to reduce their tendency to aggregation and improve colloidal stability. After incubation with G4@IONPs and subjected to an alternating magnetic field (AMF), the survival of breast cancer cells significantly reduced due to apoptosis. After intratumoral injection of G4@IONPs, tumor-bearing BALB/c mice were exposed to AMF for 20 min, three times every other day. The findings of histopathological and immunohistochemical studies did not display any metastatic cells in the liver and lung tissues in the treatment group, while the control mouse tissues contained metastatic breast cancer cells. Furthermore, magnetic hyperthermia treatment inhibited tumor growth by increasing cancer cell apoptosis and reducing tumor angiogenesis [73].In a novel study, Dheyab et al. developed gold- (Au-) coated Fe3O4 (Au-Fe3O4 NPs) for PTT to eradicate breast cancer cells (MCF7). After incubation of MCF7 cells with Au-Fe3O4 NPs for 24 h, the cell viability analysis demonstrated negligible toxicity toward cells. Significant cell reduction was observed for MCF7 (73.9%) cells following PTT at the highest concentration of NPs (Fe/ml—50 μg) for 10 min illumination at the near-infrared (NIR) region when compared with other intervention groups. It can be concluded that the synthesized Au-Fe3O4 NPs are effective and promising PTT agents for breast cancer treatment [74].


### 3.3. Drug Delivery Systems

Despite its well-known side effects, chemotherapy is one of the primary cancer treatment methods [75–78]. Additionally, many anticancer drugs have a short half-life and lose their effective concentration in the blood very soon. Furthermore, chemoresistance is a common drawback followed by chemotherapy [79, 80], a process that involves breast cancer stem-like cells (CSCs).Recently, microRNAs (miRNAs) have captured great attention since they are connected to tumorigenesis inhibition. For instance, miR-29a induces apoptosis and controls tumor invasiveness [81, 82]; however, their delivery by nanoparticles is an attractive path due to their vulnerability to nuclease degradation and imperfect absorption. Thanks to their exclusive potential, iron oxide nanoparticles can be used as carrier to deliver such therapeutic agents. In a recent study, dextran was coated on iron oxide nanoparticles to provide a hydroxyl group to allow surface attachment of miR-29a by polyethyleneimine as a linker. The results showed that this engineered drug delivery system could improve the selective delivery of miR-29a and downregulated antiapoptotic genes [83]. Baneshi et al. functionalized bovine albumin using AS1141 aptamer on a nanocarrier made from desolvation crosslinking between iron oxide and gold nanoparticles (Dox@apt-BSA IONPs-GNPs) with an average size of 120 nm. In vitro evaluation of the antitumor effect on MCF7 and SKBR3 human breast cancer cells revealed higher levels of cell death and later apoptosis for both cells. This may arise from the increased attraction of AS1411 aptamer to nucleolin-containing MCF7 plasma membrane [84]. Moreover, this multifunctional system can both photothermal and MRI imaging due to the presence of IONPs and GNPs [84].Doxorubicin is an anthracycline, which is a type of chemotherapy medication. By inhibiting an enzyme called topo isomerase2, it slows or halt cancer cell proliferation. This enzyme is required for cancer cells to divide and proliferate [85]. Doxorubicin, besides its fast clearance rate inside the body and low therapeutic index [86, 87], has some side effects on cardiac cells leading to cardiomyopathy and heart failure [88–90] and destroys bone marrow stem cells [91, 92]. Therefore, its efficient delivery is crucial. Piehler and colleagues functionalized iron oxide nanoparticles with doxorubicin and evaluated doxorubicin-induced antitumor effects in breast cancer cells (BT474) in the presence of magnetic hyperthermia (43°C, one hour). They found that intratumoral injection of doxorubicin-functionalized magnetic nanoparticles combined with magnetic hyperthermia supports better tumor regression in vivo than magnetic hyperthermia alone or intratumoral application of free doxorubicin [93]. In similar study, Askari et al. succeeded in encapsulating IONPs and doxorubicin in PEGylated liposomes via coprecipitation of iron salts and dextrose. Finally, they encapsulated the particles into the liposomes and evaluated the cytotoxicity of the synthesized agents on MCF7 cells using a lactate dehydrogenase (LDH) assay. The results confirmed that although IONPs are cytocompatible up to 50 ppm, the liposome-coated particles having the same concentration were considered toxic to the cells [94].In a recent report, 12 nm Pluronic F-127-coated magnetic particles were electrostatically attached to doxorubicin to have MRI-guided magneto-chemotherapy modality. The MCF7 cancer cell line results showed about 96% of cell death during 30 minutes of exposure to AMF of 267 kH and 300 Oe [95]. Similarly, cobalt ferrite (CoFe2O4) nanoparticles were functionalized with meso-2,3-dimercaptosuccinic acid (DMSA) to provide a binding site for doxorubicin. The outcome demonstrated the cytotoxic effect of the combination on MDA-MB-231 cancer cells [96]. In another study, a novel system was fabricated based on PEGylated magnetic nanoparticles conjugated with vascular endothelial growth factor (VEGF) antibodies to facilitate doxorubicin delivery into murine breast adenocarcinoma 4T1 cell line. Based on observations, the average survival rate of mice bearing 4T1 tumors was higher than that of the control group [97]. Szuplewska et al. located hydrophobic IONPs into the lipid bilayer and loaded them with doxorubicin. After incubating cancer and healthy cells with different concentrations of these nanomaterials, the cytotoxicity evaluation demonstrated that doxorubicin-loaded MLs selectively reduced human breast tumor cells [98]. As breast cancer progresses to the metastatic phase, many patients report the development of tumors within bones. To prevent this undesirable outcome, a combination of an anticancer drug with an antiabsorptive agent such as zoledronic acid (Zol) was investigated by Benyettou. They utilized uncoated IONPs for sequential delivery of doxorubicin and Zol to the MCF7 breast cancer cell line. First, Zol was attached to the iron oxide nanoparticles via phosphonate coordination to make Z-NPs. Finally, doxorubicin was joined to CZ-NPs to fabricate the DCZ-NP complex. The authors demonstrated that doxorubicin was released immediately after the subjection of the DCZ-NPs into the low pH solution when the particles were under magnetically induced healing and in the presence of glutathione (GSH). It was concluded that, after cell internalization, sequential delivery of doxorubicin and Zol could be effective on MCF7 death. This study, also showed that treating the MCF7 with the alternating magnetic field and DCZ-NP subjection causes a considerable increase in cell internalization and, consequently, cytotoxicity [99].Paclitaxel (PTX) is an anticancer drug administered to treat advanced solid carcinoma such as lung, ovarian, and breast cancer [100, 101]. PTX causes inhibition in the late G1 or M phases of the cell cycle to induce cell death [102–104]. Various drug delivery systems are presented to PTX targeting cancer sites. Jeon et al. fabricated a superparamagnetic iron oxide core to conjugate hydrophobic PTX and folic acid (FA) coated with short- and long-chain polyethylene glycol to improve hydrophilicity. The results confirmed that NP-PTX-FA could release the drug under conditions mimicking the acidic intracellular pH of breast cancer cells. FA caused an increase in NP cellular uptake and consequently increased cellular cytotoxicity. It was suggested that this system could represent a promising tool for delivering hydrophobic drugs [105]. In another study, Park et al. decorated iron oxide nanoparticles with PLGA and attached them with PTX to form the PTX@PINC complex. After treating orthotopic 4T1 breast tumors with PTX@PINC, it was concluded that the PTX-loaded nanoparticles were efficiently delivered to the target site via an external magnetic field and assisted the tumor and metastasis inhibition [106]. Although numerous nanoparticles have been developed to deliver PTX to target sites, achieve sufficient drug loading, and have a small particle size, maintaining nanoparticle stability remains a challenge.Methotrexate (MTX) is another anticancer agent that interferes with cellular folate metabolism through specific mechanisms and causes cancer cell death [107]. Also, this molecule facilitates nanocarrier’s internalization within the cancer cells [108]. MTX is helpful in many malignancies, such as leukemia, hematologic malignancies, osteosarcoma, breast and cervical cancer, and even rheumatoid arthritis [109]. Nosrati et al. designed and fabricated a drug delivery system via conjugation of MTX on the surface of L-lysine-coated iron oxide magnetic nanoparticles (F-Lys-MTX NPs). It was concluded that F-Lys-MTX NPs could be an excellent system against breast cancer cell lines [110]. In a similar study, IONPs were coated with arginine before conjugation with MTX, termed Fe-Arg-MTX. MTX was attached to the surface of iron oxide nanoparticles via bound between its carboxylic acid groups and arginine groups. MTX was cleaved from carriers in the presence of protease-like lysosomal conditions. Results revealed that uncoated IONPs presented no cytotoxic effects against MCF7, 4T1, and HFF-2 cell lines, whereas Fe-Arg-MTX caused a significant decrease in cancer cell viability [106].Some agents can be exploited to block the overexpressed receptor on breast cancer cells such as human epithermal growth factor receptors which are classified as the HER2-positive (HER2+) subtype. For this purpose, simultaneous application of conventional chemotherapies with particular drugs such as lapatinib, pertuzumab, and trastuzumab emtansine has been suggested. The anti-HER2 monoclonal antibody trastuzumab represents the gold standard for the treatment of HER2+ breast cancer patients [111, 112]. Truffi et al. developed a nanosystem based on the conjugation of trastuzumab onto the magnetic iron oxide nanoparticles (MNP-HC) to treat HER2+ patients. Their findings indicated that the attached trastuzumab on the nanoparticle surface increased anticancer efficacy in HER2+ breast cancer cells [113]. In another study, small interfering RNA (siRNA) was applied to HER2+ breast cancer patients. Accordingly, magnetic iron oxide nanoparticles were coated with caffeic acid and stabilized by layers of calcium phosphate and PEG-polyanion block copolymer to incorporate siRNA. Also, the fabricated drug delivery system was successfully tacked up by HER2+ breast cancer cell line HCC1954 that enhanced under magnetic guidance. Results demonstrated that nanoparticles skipped from endosomal degradation and delivered siRNA into the cytoplasm, inducing HER2 gene silencing (Figure 4) [114].Figure 4
Procedures using iron oxide nanoparticles (IONPs) in combating breast cancer. IONPs can be exploited in different forms; such as core elements of nanoparticles or in the lipid bilayer of magnetoliposomes. (a) iWnt-ATF24-IONP-Dox is designed to deliver doxorubicin (Dox) to Dox-resistant tumors. Targeting both urokinase plasminogen activator receptor (uPAR) and Wnt/LRP5/6 receptors improves the antitumor properties of nanoparticles and inhibits invasion of cancer cells. (b) Magnetoliposomes are Zin the selected delivery of anticancer drugs. Superparamagnetic iron oxide nanoparticles (SPIONs) can be used inside or between the lipid bilayers. Targeting tumor cells using a magnetic field causes vibration in the SPIONs, and consequently, the lipid bilayer becomes unstable. In this condition, loaded drugs can be released and selectively combat the tumor cells. (c) Trastuzumab is an anti-human epidermal growth factor receptor 2 (HER2) antibody that is useful in the treatment of HER2+ breast cancer patients. In the MNP-HC, half-chains of trastuzumab are attached to the IONPs to enhance the antitumor characteristics of the drug. (d) Small interfering RNA (siRNA) therapy is another approach to combat HER2+ tumors. Some nanoparticles such as Caf-MCaP are designed to deliver siRNAs into the cancer cells. Caf-MCaP enters the cell through an energy-dependent manner (like pinocytosis) and decomposes in the acidic pH of endosomes or lysosomes. Other contents of this nanoparticle help the siRNAs to escape the vesicle and enter the cytoplasm.In addition to conventional drugs, herbal-based components have attracted great deal of attraction for application in the field of breast cancer treatment using iron oxide nanoparticles.Syzygium aromaticum (clove) is an example of herbal-based bioactive molecule which represents anticarcinogenic, antimutagenic, aphrodisiac, antimicrobial, antioxidant, and anti-inflammatory properties. Cloves have been acclaimed as the cancer therapy of the future due to their capacity to trigger apoptosis and function in a variety of cancer cells [115]. Cloves also contain betulinic acid and other triterpenes, which can help prevent breast cancer by acting as chemopreventive agents [116]. In an experiment, iron oxide nanoparticles were functionalized with clove to induce MCF7 breast cancer cell apoptosis. Iron nanoparticles were synthesized through the coprecipitation method, coated with polyvinylpyrrolidone (PVP), and loaded with Syzygium aromaticum. Flow cytometry analysis proved that the percentage of cells in terms of viable early and late apoptosis was increased. Moreover, the cell cycle analysis showed a substantial accumulation of nanoparticles in the sub-G1 phase, establishing apoptosis induction. In addition, enhanced activities of caspase-3, caspase-8, and caspase-9 suggested that IONPs could trigger caspase activity. Besides, functionalized nanoparticles induced oxidative stress through reactive oxygen species (ROS) formation [117].


## 3.1. Imaging Modalities

### 3.1.1. MRI

Early detection of breast cancer, which is crucial to limit progression to the metastatic phase, is routinely achieved using mammography, ultrasound, and MRI [34, 35]. MRI takes advantage of protons’ magnetic properties to make high-resolution images from different body parts when patients are situated in a magnetic field. However, this technique might give false positives, especially at ductal carcinoma in situ (DCIS) diagnosis [36, 37]. Han et al. have developed a method to synthesize magnetic-fluorescent iron oxide-carbon hybrid nanomaterials using a one-pot reaction. These nanoparticles not only have many magnetic features for cancer cell detection but also; thanks to their fluorescent characteristics, colorful imaging of cells is possible as well [38].Moreover, since a cluster of differentiation 44 (CD44) is usually overexpressed in aggressive breast cancer cells [39, 40], the hybrid nanoparticles were conjugated with CD44 monoclonal antibodies to provide fluorescent/MRIs with an enhanced track of cancer cells [38]. MUC1, a compound involved in cell-cell and cell-matrix interaction [41], is another overexpressed glycoprotein in adenocarcinomas such as breast cancer cells [42]. Relying on this fact, Khaniabadi and coworkers have detailed the development of a magnetic contrast agent comprised of superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with C595 monoclonal antibody to target MUC1 overexpressed MCF7 cells. These nanoprobes showed excellent biocompatibility with low cytotoxicity. In addition, magnetic resonance contrast agents, at high concentration, decrease T1 and T2 relaxation times, which enhance MRI efficacy [43].Rezayan et al. reported surface modification of Fe3O4 nanoparticles with polyethylene glycol diacid and dopamine as a linker to increase the biocompatibility and dispersity of the particles. Various concentrations and time points were examined to assess the efficacy of the nanoparticles for MRI application. The results demonstrated a strong reverse relationship between the agents’ concentration and relaxation time, leading to improved magnetic resonance signal. Moreover, MTT assays on MDA-MB-231 cancer cells were performed at various concentrations of modified and unmodified nanoparticles for 24 and 48 hours. The results demonstrated that the PEGylated IONPs were less cytotoxic due to the less Fe ion release from particles and lower chemical activity. The cellular absorption of coated nanoparticles was 80% comparing to the unmodified ones with 9% [44].


### 3.1.2. Computerized Tomography (CT)

CT applies X-ray to provide two-dimensional images of the body, and different techniques are used to make three-dimensional images to detect the site of the lesion. However, this method lacks a desirable resolution and needs an intravenous injection of a contrast agent to enhance the image quality [45]. To provide high-resolution and high-sensitive images, Deng et al. synthesized a novel combination of ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) with cyclic arginine-glycine-aspartate (cRGD) and radiolabeled to use as bimodal imaging agents for single-photon emission computerized tomography (SPECT) and MRI [46]. Since αVβ3 receptors are overexpressed in cancer and endothelial cells in breast cancer [47], cRGD was used due to its high affinity to these receptors. They were coated with carboxymethyl dextran to modify the nanoparticles, conjugated with cRGD peptides via the carboxyl groups on the surface, radiolabeled with iodine. These radiolabeled nanoparticles had high tumor absorption, moderate liver elimination, and long circulation half-life. However, iodine was not entirely appropriate as a radioisotope component due to low gamma-ray emission [46].


### 3.1.3. Combined Imaging Approaches

Imaging techniques have been integrated in recent years to provide an optimal balance of sensitivity and specificity, as well as superior quality in cancer diagnosis [48, 49]. Using this combination, many researches have been performed to investigate the application of iron oxide nanoparticles in the detection of breast cancer. For instance, in a study, Thin et al. introduced a novel combination imaging technique of MRI, SPECT, and bioluminescence imaging (BLI) with adipocyte-derived mesenchymal stem cells (ADSCs) [50]. Mesenchymal stem cells have shown great potential for the treatment of numerous diseases [51], particularly as an assistive system to integrate tumors, as they are easily extracted from various sources and can be genetically manipulated [52]. The ADSCs were radiolabeled with indium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid- (DOTA-) SPIONs and injected both intravenously and intracardially into the mice with 4T1 breast tumor. This study shed light on the importance of stem cell therapy and stem cell tracking in vivo.A study by Kanwar et al. evaluated antitumor and imaging characteristics of Fe3O4-saturated bovine lactoferrin nanocapsules (FebLf NCs) in BALB/c nude mouse xenograft model. The results demonstrated that the FebLf NCs were absorbed into the breast cancer cells via the surface receptors (LRP1, LRP2, TFR-1, and TFR-2) and induced cell death. Moreover, they provided near-infrared images demonstrating the efficacy of these particles in tumor growth control [53]. In a similar study, de Souza Albernaz and colleagues produced IONPs coupled with diethylenetriaminepentaacetic acid (DTPA) and trastuzumab. They labeled them with two radioisotopes of Gallium-68 and Technetium-99 to conduct PET and SPECT imaging. In vivo evaluation in 6-week BALB/c nude mice showed that more than 80% of the dual-modality agents were removed in healthy mice via the renal system, while in the xenograft mice, the tumors internalized a considerable amount of 1.98 μCi due to their HER2 overexpression [54].To track anticancer drugs in a shorter time and a noninvasive manner, Wang et al. reported synthesizing fluorine-18-labeled arginine–glycine–aspartic acid- (RGD-) coupled ultrasmall iron oxide nanoparticle (18F-RGD@USPIO) using click chemistry. They utilized PET/CT and MRI techniques to monitor the antiangiogenic influence of intraperitoneally injected bevacizumab anticancer drugs over seven days. In this study, in vitro testing was conducted on MDA-MB-231 cell lines, and in vivo experiments were conducted using MDA-MB-231 xenograft-bearing mouse models. The results proved high stability, low cytotoxicity, and excellent biodistribution of the agents. Moreover, PET/CT and MRI imaging confirmed a lower 18F-RGD@USPIO coupling after bevacizumab injection due to the tumor’s low vascular density [55].


## 3.1.1. MRI

Early detection of breast cancer, which is crucial to limit progression to the metastatic phase, is routinely achieved using mammography, ultrasound, and MRI [34, 35]. MRI takes advantage of protons’ magnetic properties to make high-resolution images from different body parts when patients are situated in a magnetic field. However, this technique might give false positives, especially at ductal carcinoma in situ (DCIS) diagnosis [36, 37]. Han et al. have developed a method to synthesize magnetic-fluorescent iron oxide-carbon hybrid nanomaterials using a one-pot reaction. These nanoparticles not only have many magnetic features for cancer cell detection but also; thanks to their fluorescent characteristics, colorful imaging of cells is possible as well [38].Moreover, since a cluster of differentiation 44 (CD44) is usually overexpressed in aggressive breast cancer cells [39, 40], the hybrid nanoparticles were conjugated with CD44 monoclonal antibodies to provide fluorescent/MRIs with an enhanced track of cancer cells [38]. MUC1, a compound involved in cell-cell and cell-matrix interaction [41], is another overexpressed glycoprotein in adenocarcinomas such as breast cancer cells [42]. Relying on this fact, Khaniabadi and coworkers have detailed the development of a magnetic contrast agent comprised of superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with C595 monoclonal antibody to target MUC1 overexpressed MCF7 cells. These nanoprobes showed excellent biocompatibility with low cytotoxicity. In addition, magnetic resonance contrast agents, at high concentration, decrease T1 and T2 relaxation times, which enhance MRI efficacy [43].Rezayan et al. reported surface modification of Fe3O4 nanoparticles with polyethylene glycol diacid and dopamine as a linker to increase the biocompatibility and dispersity of the particles. Various concentrations and time points were examined to assess the efficacy of the nanoparticles for MRI application. The results demonstrated a strong reverse relationship between the agents’ concentration and relaxation time, leading to improved magnetic resonance signal. Moreover, MTT assays on MDA-MB-231 cancer cells were performed at various concentrations of modified and unmodified nanoparticles for 24 and 48 hours. The results demonstrated that the PEGylated IONPs were less cytotoxic due to the less Fe ion release from particles and lower chemical activity. The cellular absorption of coated nanoparticles was 80% comparing to the unmodified ones with 9% [44].


## 3.1.2. Computerized Tomography (CT)

CT applies X-ray to provide two-dimensional images of the body, and different techniques are used to make three-dimensional images to detect the site of the lesion. However, this method lacks a desirable resolution and needs an intravenous injection of a contrast agent to enhance the image quality [45]. To provide high-resolution and high-sensitive images, Deng et al. synthesized a novel combination of ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) with cyclic arginine-glycine-aspartate (cRGD) and radiolabeled to use as bimodal imaging agents for single-photon emission computerized tomography (SPECT) and MRI [46]. Since αVβ3 receptors are overexpressed in cancer and endothelial cells in breast cancer [47], cRGD was used due to its high affinity to these receptors. They were coated with carboxymethyl dextran to modify the nanoparticles, conjugated with cRGD peptides via the carboxyl groups on the surface, radiolabeled with iodine. These radiolabeled nanoparticles had high tumor absorption, moderate liver elimination, and long circulation half-life. However, iodine was not entirely appropriate as a radioisotope component due to low gamma-ray emission [46].


## 3.1.3. Combined Imaging Approaches

Imaging techniques have been integrated in recent years to provide an optimal balance of sensitivity and specificity, as well as superior quality in cancer diagnosis [48, 49]. Using this combination, many researches have been performed to investigate the application of iron oxide nanoparticles in the detection of breast cancer. For instance, in a study, Thin et al. introduced a novel combination imaging technique of MRI, SPECT, and bioluminescence imaging (BLI) with adipocyte-derived mesenchymal stem cells (ADSCs) [50]. Mesenchymal stem cells have shown great potential for the treatment of numerous diseases [51], particularly as an assistive system to integrate tumors, as they are easily extracted from various sources and can be genetically manipulated [52]. The ADSCs were radiolabeled with indium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid- (DOTA-) SPIONs and injected both intravenously and intracardially into the mice with 4T1 breast tumor. This study shed light on the importance of stem cell therapy and stem cell tracking in vivo.A study by Kanwar et al. evaluated antitumor and imaging characteristics of Fe3O4-saturated bovine lactoferrin nanocapsules (FebLf NCs) in BALB/c nude mouse xenograft model. The results demonstrated that the FebLf NCs were absorbed into the breast cancer cells via the surface receptors (LRP1, LRP2, TFR-1, and TFR-2) and induced cell death. Moreover, they provided near-infrared images demonstrating the efficacy of these particles in tumor growth control [53]. In a similar study, de Souza Albernaz and colleagues produced IONPs coupled with diethylenetriaminepentaacetic acid (DTPA) and trastuzumab. They labeled them with two radioisotopes of Gallium-68 and Technetium-99 to conduct PET and SPECT imaging. In vivo evaluation in 6-week BALB/c nude mice showed that more than 80% of the dual-modality agents were removed in healthy mice via the renal system, while in the xenograft mice, the tumors internalized a considerable amount of 1.98 μCi due to their HER2 overexpression [54].To track anticancer drugs in a shorter time and a noninvasive manner, Wang et al. reported synthesizing fluorine-18-labeled arginine–glycine–aspartic acid- (RGD-) coupled ultrasmall iron oxide nanoparticle (18F-RGD@USPIO) using click chemistry. They utilized PET/CT and MRI techniques to monitor the antiangiogenic influence of intraperitoneally injected bevacizumab anticancer drugs over seven days. In this study, in vitro testing was conducted on MDA-MB-231 cell lines, and in vivo experiments were conducted using MDA-MB-231 xenograft-bearing mouse models. The results proved high stability, low cytotoxicity, and excellent biodistribution of the agents. Moreover, PET/CT and MRI imaging confirmed a lower 18F-RGD@USPIO coupling after bevacizumab injection due to the tumor’s low vascular density [55].


## 3.2. Magnetic Hyperthermia

Magnetic hyperthermia is a therapeutic process by which the internal temperature of cancer-affected tissues is raised to 43–46°C to kill cancer cells directly [56]. It is administered together with other cancer treatment modalities to control tumor growth [57]. The application of iron nanoparticles can avoid damaging the surrounding tissue since they help increase the temperature in the nanoscale within the targeted tissue [56]. Magnetic nanoparticles can generate heat inside a tumor when placed in an alternating magnetic field (AMF) via Neel and Brownian mechanisms [58]. Hyperthermia executes cancer cells via various paths such as necrosis, apoptosis, protein denaturation, and immune system reaction to heat shock proteins produced due to the stress on cancer cells [56, 59, 60]. Recently the application of iron oxide nanoparticles for hyperthermia has received much attention [61–64]. Munir et al. reported the coating of IONPs with malic and citric acid, which resulted in increased magnetic polarization and dimension reduction. After injecting the modified nanoparticles into the breast tumor in the mice, the animal models were exposed to a 15 mT magnetic field and frequency of 100 kHz for one hour. The histological analysis indicated that the AMF caused apoptosis of the cancer cells in the tumor [65]. Cędrowska et al. reported a novel conjugation of trastuzumab monoclonal antibody (Herceptin commercial drug) on SPIONs using 3-phosphonopropionic acid (CEPA) and Actinium-225 isotope as an emitter. The 225Ac@Fe3O4-CEPA-trastuzumab nanoparticles can specifically link to HER2 receptors which are overexpressed on the surface of the breast and ovarian cancer cells. It has been observed that these nanoparticles could target cancer cells and represented radiotherapy and hyperthermia functions [66]. Zuvin et al. developed poly (acrylic acid)-coated USPIONs, labeled with anti-HER2 antibodies to target breast cancer cells, and exposed them to a magnetic strength as low as 0.8 kAm-1 preventing eddy current, which can have detrimental effects on normal tissues. Following in vitro evaluation, it was demonstrated that although the synthesized nanoparticles were not cytotoxic, a considerable reduction in cell proliferation and cell death was spotted among MDA-MB-453 cancer cells after exposure to the magnetic field [67]. In a similar study by Almaki et al., core-shell magnetic nanoparticles coated with polyethylene glycol (PEG) with an average size of less than 150 nm were produced, and trastuzumab was immobilized on the surface of the nanoparticles. To investigate hyperthermia treatment in vitro, MTT assays were performed on different cell lines showing nontoxicity. The results revealed nanoparticle accumulation within the tumor at levels four times higher than the level of accumulation within normal tissues, confirming the considerable potential of the magnetic tumor-targeting hyperthermia concept for breast cancer treatment [68]. A variety of mammalian cells, including cancer cells, express CD-44 antigen, a glycoprotein responsible for different cell behavior, such as proliferation, differentiation, and cell adhesion [69]. In a recent report, hyaluronic acid that is a ligand for CD-44 was covalently immobilized on graphene oxide particles; moreover, doxorubicin and paclitaxel as chemo drugs were added separately to this combination to provide drug-targeting agents. Additionally, to provide hyperthermia functionality, iron oxide nanoparticles were inserted and composited with them. The cytotoxicity assay on MDA-MB-231 and BT-474 breast cancer cell lines proved the high cytotoxic effect on MDA-MB-231 cells with CD44 expression, but not on BT-474 cells having not CD44 [70]. In another interesting study, multifunctional Off-On nanoparticles were synthesized using IONPs as nanocores and poly (lactic-co-glycolic acid) (PLGA) as the nanoshell. In this study, an anticancer drug was packed between core and shell, and polyvinylpyrrolidone (PVP) was added to the core-shell particles. This pH-responsive structure is in the Off mode when circulating inside the body and in the On mode when they enter the cells and places them in an acidic condition causing drug releases. The hyperthermia contributed to suppressing cell growth and increasing the temperature from 37 to 42°C leading to a faster PLGA degradation causing more tamoxifen release and MCF7 apoptosis [71].In a combinational therapy, iron oxide nanoparticles were coated with gold (Au@IONPs) and exposed to radiofrequency (RF) waves and X-ray radiation to provide both RF hyperthermia and radiotherapy for MCF7 apoptosis. The results showed a significantly improved radiotherapy effect when accompanied by RF hyperthermia [72]. In an experiment conducted by Salimi et al., iron oxide nanoparticles were used for magnetic hyperthermia treatment of breast cancer in BALB/c mice. In this study, the IONPs were functionalized with the fourth generation (G4) of poly amidoamine (PAMAM) dendrimers (G4@IONPs) to reduce their tendency to aggregation and improve colloidal stability. After incubation with G4@IONPs and subjected to an alternating magnetic field (AMF), the survival of breast cancer cells significantly reduced due to apoptosis. After intratumoral injection of G4@IONPs, tumor-bearing BALB/c mice were exposed to AMF for 20 min, three times every other day. The findings of histopathological and immunohistochemical studies did not display any metastatic cells in the liver and lung tissues in the treatment group, while the control mouse tissues contained metastatic breast cancer cells. Furthermore, magnetic hyperthermia treatment inhibited tumor growth by increasing cancer cell apoptosis and reducing tumor angiogenesis [73].In a novel study, Dheyab et al. developed gold- (Au-) coated Fe3O4 (Au-Fe3O4 NPs) for PTT to eradicate breast cancer cells (MCF7). After incubation of MCF7 cells with Au-Fe3O4 NPs for 24 h, the cell viability analysis demonstrated negligible toxicity toward cells. Significant cell reduction was observed for MCF7 (73.9%) cells following PTT at the highest concentration of NPs (Fe/ml—50 μg) for 10 min illumination at the near-infrared (NIR) region when compared with other intervention groups. It can be concluded that the synthesized Au-Fe3O4 NPs are effective and promising PTT agents for breast cancer treatment [74].


## 3.3. Drug Delivery Systems

Despite its well-known side effects, chemotherapy is one of the primary cancer treatment methods [75–78]. Additionally, many anticancer drugs have a short half-life and lose their effective concentration in the blood very soon. Furthermore, chemoresistance is a common drawback followed by chemotherapy [79, 80], a process that involves breast cancer stem-like cells (CSCs).Recently, microRNAs (miRNAs) have captured great attention since they are connected to tumorigenesis inhibition. For instance, miR-29a induces apoptosis and controls tumor invasiveness [81, 82]; however, their delivery by nanoparticles is an attractive path due to their vulnerability to nuclease degradation and imperfect absorption. Thanks to their exclusive potential, iron oxide nanoparticles can be used as carrier to deliver such therapeutic agents. In a recent study, dextran was coated on iron oxide nanoparticles to provide a hydroxyl group to allow surface attachment of miR-29a by polyethyleneimine as a linker. The results showed that this engineered drug delivery system could improve the selective delivery of miR-29a and downregulated antiapoptotic genes [83]. Baneshi et al. functionalized bovine albumin using AS1141 aptamer on a nanocarrier made from desolvation crosslinking between iron oxide and gold nanoparticles (Dox@apt-BSA IONPs-GNPs) with an average size of 120 nm. In vitro evaluation of the antitumor effect on MCF7 and SKBR3 human breast cancer cells revealed higher levels of cell death and later apoptosis for both cells. This may arise from the increased attraction of AS1411 aptamer to nucleolin-containing MCF7 plasma membrane [84]. Moreover, this multifunctional system can both photothermal and MRI imaging due to the presence of IONPs and GNPs [84].Doxorubicin is an anthracycline, which is a type of chemotherapy medication. By inhibiting an enzyme called topo isomerase2, it slows or halt cancer cell proliferation. This enzyme is required for cancer cells to divide and proliferate [85]. Doxorubicin, besides its fast clearance rate inside the body and low therapeutic index [86, 87], has some side effects on cardiac cells leading to cardiomyopathy and heart failure [88–90] and destroys bone marrow stem cells [91, 92]. Therefore, its efficient delivery is crucial. Piehler and colleagues functionalized iron oxide nanoparticles with doxorubicin and evaluated doxorubicin-induced antitumor effects in breast cancer cells (BT474) in the presence of magnetic hyperthermia (43°C, one hour). They found that intratumoral injection of doxorubicin-functionalized magnetic nanoparticles combined with magnetic hyperthermia supports better tumor regression in vivo than magnetic hyperthermia alone or intratumoral application of free doxorubicin [93]. In similar study, Askari et al. succeeded in encapsulating IONPs and doxorubicin in PEGylated liposomes via coprecipitation of iron salts and dextrose. Finally, they encapsulated the particles into the liposomes and evaluated the cytotoxicity of the synthesized agents on MCF7 cells using a lactate dehydrogenase (LDH) assay. The results confirmed that although IONPs are cytocompatible up to 50 ppm, the liposome-coated particles having the same concentration were considered toxic to the cells [94].In a recent report, 12 nm Pluronic F-127-coated magnetic particles were electrostatically attached to doxorubicin to have MRI-guided magneto-chemotherapy modality. The MCF7 cancer cell line results showed about 96% of cell death during 30 minutes of exposure to AMF of 267 kH and 300 Oe [95]. Similarly, cobalt ferrite (CoFe2O4) nanoparticles were functionalized with meso-2,3-dimercaptosuccinic acid (DMSA) to provide a binding site for doxorubicin. The outcome demonstrated the cytotoxic effect of the combination on MDA-MB-231 cancer cells [96]. In another study, a novel system was fabricated based on PEGylated magnetic nanoparticles conjugated with vascular endothelial growth factor (VEGF) antibodies to facilitate doxorubicin delivery into murine breast adenocarcinoma 4T1 cell line. Based on observations, the average survival rate of mice bearing 4T1 tumors was higher than that of the control group [97]. Szuplewska et al. located hydrophobic IONPs into the lipid bilayer and loaded them with doxorubicin. After incubating cancer and healthy cells with different concentrations of these nanomaterials, the cytotoxicity evaluation demonstrated that doxorubicin-loaded MLs selectively reduced human breast tumor cells [98]. As breast cancer progresses to the metastatic phase, many patients report the development of tumors within bones. To prevent this undesirable outcome, a combination of an anticancer drug with an antiabsorptive agent such as zoledronic acid (Zol) was investigated by Benyettou. They utilized uncoated IONPs for sequential delivery of doxorubicin and Zol to the MCF7 breast cancer cell line. First, Zol was attached to the iron oxide nanoparticles via phosphonate coordination to make Z-NPs. Finally, doxorubicin was joined to CZ-NPs to fabricate the DCZ-NP complex. The authors demonstrated that doxorubicin was released immediately after the subjection of the DCZ-NPs into the low pH solution when the particles were under magnetically induced healing and in the presence of glutathione (GSH). It was concluded that, after cell internalization, sequential delivery of doxorubicin and Zol could be effective on MCF7 death. This study, also showed that treating the MCF7 with the alternating magnetic field and DCZ-NP subjection causes a considerable increase in cell internalization and, consequently, cytotoxicity [99].Paclitaxel (PTX) is an anticancer drug administered to treat advanced solid carcinoma such as lung, ovarian, and breast cancer [100, 101]. PTX causes inhibition in the late G1 or M phases of the cell cycle to induce cell death [102–104]. Various drug delivery systems are presented to PTX targeting cancer sites. Jeon et al. fabricated a superparamagnetic iron oxide core to conjugate hydrophobic PTX and folic acid (FA) coated with short- and long-chain polyethylene glycol to improve hydrophilicity. The results confirmed that NP-PTX-FA could release the drug under conditions mimicking the acidic intracellular pH of breast cancer cells. FA caused an increase in NP cellular uptake and consequently increased cellular cytotoxicity. It was suggested that this system could represent a promising tool for delivering hydrophobic drugs [105]. In another study, Park et al. decorated iron oxide nanoparticles with PLGA and attached them with PTX to form the PTX@PINC complex. After treating orthotopic 4T1 breast tumors with PTX@PINC, it was concluded that the PTX-loaded nanoparticles were efficiently delivered to the target site via an external magnetic field and assisted the tumor and metastasis inhibition [106]. Although numerous nanoparticles have been developed to deliver PTX to target sites, achieve sufficient drug loading, and have a small particle size, maintaining nanoparticle stability remains a challenge.Methotrexate (MTX) is another anticancer agent that interferes with cellular folate metabolism through specific mechanisms and causes cancer cell death [107]. Also, this molecule facilitates nanocarrier’s internalization within the cancer cells [108]. MTX is helpful in many malignancies, such as leukemia, hematologic malignancies, osteosarcoma, breast and cervical cancer, and even rheumatoid arthritis [109]. Nosrati et al. designed and fabricated a drug delivery system via conjugation of MTX on the surface of L-lysine-coated iron oxide magnetic nanoparticles (F-Lys-MTX NPs). It was concluded that F-Lys-MTX NPs could be an excellent system against breast cancer cell lines [110]. In a similar study, IONPs were coated with arginine before conjugation with MTX, termed Fe-Arg-MTX. MTX was attached to the surface of iron oxide nanoparticles via bound between its carboxylic acid groups and arginine groups. MTX was cleaved from carriers in the presence of protease-like lysosomal conditions. Results revealed that uncoated IONPs presented no cytotoxic effects against MCF7, 4T1, and HFF-2 cell lines, whereas Fe-Arg-MTX caused a significant decrease in cancer cell viability [106].Some agents can be exploited to block the overexpressed receptor on breast cancer cells such as human epithermal growth factor receptors which are classified as the HER2-positive (HER2+) subtype. For this purpose, simultaneous application of conventional chemotherapies with particular drugs such as lapatinib, pertuzumab, and trastuzumab emtansine has been suggested. The anti-HER2 monoclonal antibody trastuzumab represents the gold standard for the treatment of HER2+ breast cancer patients [111, 112]. Truffi et al. developed a nanosystem based on the conjugation of trastuzumab onto the magnetic iron oxide nanoparticles (MNP-HC) to treat HER2+ patients. Their findings indicated that the attached trastuzumab on the nanoparticle surface increased anticancer efficacy in HER2+ breast cancer cells [113]. In another study, small interfering RNA (siRNA) was applied to HER2+ breast cancer patients. Accordingly, magnetic iron oxide nanoparticles were coated with caffeic acid and stabilized by layers of calcium phosphate and PEG-polyanion block copolymer to incorporate siRNA. Also, the fabricated drug delivery system was successfully tacked up by HER2+ breast cancer cell line HCC1954 that enhanced under magnetic guidance. Results demonstrated that nanoparticles skipped from endosomal degradation and delivered siRNA into the cytoplasm, inducing HER2 gene silencing (Figure 4) [114].Figure 4
Procedures using iron oxide nanoparticles (IONPs) in combating breast cancer. IONPs can be exploited in different forms; such as core elements of nanoparticles or in the lipid bilayer of magnetoliposomes. (a) iWnt-ATF24-IONP-Dox is designed to deliver doxorubicin (Dox) to Dox-resistant tumors. Targeting both urokinase plasminogen activator receptor (uPAR) and Wnt/LRP5/6 receptors improves the antitumor properties of nanoparticles and inhibits invasion of cancer cells. (b) Magnetoliposomes are Zin the selected delivery of anticancer drugs. Superparamagnetic iron oxide nanoparticles (SPIONs) can be used inside or between the lipid bilayers. Targeting tumor cells using a magnetic field causes vibration in the SPIONs, and consequently, the lipid bilayer becomes unstable. In this condition, loaded drugs can be released and selectively combat the tumor cells. (c) Trastuzumab is an anti-human epidermal growth factor receptor 2 (HER2) antibody that is useful in the treatment of HER2+ breast cancer patients. In the MNP-HC, half-chains of trastuzumab are attached to the IONPs to enhance the antitumor characteristics of the drug. (d) Small interfering RNA (siRNA) therapy is another approach to combat HER2+ tumors. Some nanoparticles such as Caf-MCaP are designed to deliver siRNAs into the cancer cells. Caf-MCaP enters the cell through an energy-dependent manner (like pinocytosis) and decomposes in the acidic pH of endosomes or lysosomes. Other contents of this nanoparticle help the siRNAs to escape the vesicle and enter the cytoplasm.In addition to conventional drugs, herbal-based components have attracted great deal of attraction for application in the field of breast cancer treatment using iron oxide nanoparticles.Syzygium aromaticum (clove) is an example of herbal-based bioactive molecule which represents anticarcinogenic, antimutagenic, aphrodisiac, antimicrobial, antioxidant, and anti-inflammatory properties. Cloves have been acclaimed as the cancer therapy of the future due to their capacity to trigger apoptosis and function in a variety of cancer cells [115]. Cloves also contain betulinic acid and other triterpenes, which can help prevent breast cancer by acting as chemopreventive agents [116]. In an experiment, iron oxide nanoparticles were functionalized with clove to induce MCF7 breast cancer cell apoptosis. Iron nanoparticles were synthesized through the coprecipitation method, coated with polyvinylpyrrolidone (PVP), and loaded with Syzygium aromaticum. Flow cytometry analysis proved that the percentage of cells in terms of viable early and late apoptosis was increased. Moreover, the cell cycle analysis showed a substantial accumulation of nanoparticles in the sub-G1 phase, establishing apoptosis induction. In addition, enhanced activities of caspase-3, caspase-8, and caspase-9 suggested that IONPs could trigger caspase activity. Besides, functionalized nanoparticles induced oxidative stress through reactive oxygen species (ROS) formation [117].


## 4. Future Perspective

Nanotechnology has been noticeably employed to detect and treat various diseases and has led to numerous medical developments. This review showed the potential use of iron oxide nanoparticles as therapeutic agents and acknowledged their merit over conventional therapeutic agents. Although IONPs are only commercialized to treat iron-deficient anemia and cancer treatment, several experiments are being performed to promote their clinical applications. For instance, some methods are being evaluated to synthesize pure and stable IONPs with higher production yields that do not contain toxic by-products. Also, iron nanoparticles can be modified to improve diagnostic sensitivity and specificity to make significant advances in personalized medicine.Moreover, an external demand source of affordable energy to activate IONPs located in the targeted organ is being developed to inhibit their side effects in healthy tissues. Some critical experiments are being carried out for anticancer drug targeting. For example, functionalizing IONPs with specific molecules preferentially activate specific functional groups in a target protein to change genes and related proteins’ signatures. In such approaches, nanoparticle stability and drug resistance are essential in the targeting mechanism.On the other hand, specific methods such as conjugation of breast cancer-targeting antibodies on the IONPs’ surface are recently developed to detect breast tumors. Through normal blood flow or local injection, these modified nanoparticles could bind to the cells that facilitate the detection of the cancer cells. For example, MagSense™ technology exploited this procedure for breast cancer identification. The biofunctionalized nanoparticles detect actual tumor cells, and unlike mammograms or ultrasound, the signals are not influenced by the surrounding tissues. Moreover, these nanoparticles need no radiation and eliminate the need for unnecessary biopsy procedures. Nanoparticles are popular due to the development of nanotechnology-related knowledge and research. Only a handful of them, however, make it to clinical trials. Although every nanoparticle has unique properties in the in vitro stage, they encounter comparable biological, technical, and societal challenges that hamper clinical translation [118]. Also, NPs are usually administered intravenously, so they cannot remain in the target site and interact with it. Consequently, a high concentration of them is used, which may not result in therapeutic benefits [119]. In addition, controlling the biological function of nanoparticles is very difficult and time-consuming despite introducing biosafety materials to improve nanoparticles’ retention length and half-life [3].There are some NPs’ technical challenges, such as scale-up synthesis and equal optimization. Each of these is imperative to ensure the successful clinical implementation of NPs. Most nanoparticles are made in small batches, and scaling to large amounts may not always be feasible due to technical factors. There is no systematic planning and optimization of the best-performing clinical candidates in animal models [120–122].In human trials, it is difficult to predict the effectiveness and performance of nanoparticles, and it is nearly impossible to reproduce in vivo results. Simulation of physiological tissue and its environment using computational or theoretical models can be achieved in conjunction with experimental data [118]. The majority of the research is based on “cell and animal models,” which may or may not provide understandable outcomes in human trials. As a result, simulating spontaneous responses in the human body with a single model is challenging [123]. It is expected that more clinical trials on critical issues can be addressed by applying iron nanoparticles for breast cancer diagnosis and treatment.



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