Recent Progress in Superparamagnetic Iron Oxide Nanoparticles

Qi NIE, Jian WEN

Abstract Superparamagnetic iron oxide nanoparticles （SPIONs） have immeasurable potentials in many fields such as nanobiotechnology and biomedical engineering because of their superparamagnetic properties and small particle size. This review introduces the methods for SPIONs synthesis， including co-precipitation， thermal decomposition， microemulsion and hydrothermal reaction， and surface modification of SPIONs with organometallic and inorganic metals， surface modification for targeted drug delivery， and the use of SPIONs as a contrast agent. In addition， this article also provides an overview of recent progress in SPIONs for the treatment of glioma， lung cancer and breast cancer.

Key words Superparamagnetic iron oxide nanoparticles; Tumor therapy; Synthesis; Surface modification; Contrast agent

Received： December 26， 2022  Accepted： February 27， 2023

Supported by National Natural Science Foundation of China （32060228）.

Qi NIE （1996-）， female， P. R. China， a postgraduate student majoring in pharmacy， E-mail： nq15185275337@163.com.

*Corresponding author. E-mail： wenjian2400@163.com.

Cancer， an abnormal mutation of cells， with immense potential to spread to other organs， has long been a global health concern［1］. In fact， cancer refers to more than 100 forms of the disease. In 2020， over 19 million people worldwide were diagnosed with cancer and nearly 10 million died from it. By 2040， the total number of new cancer cases will grow to approximately 28 million， and 16 million will die from it［2］. The environment is one of the most important factors influencing the development of cancer， including environmental pollutants， carcinogens and mutagens， bacterial and viral infections， and genetic susceptibility［3-4］. Depending on the cancer type， stage and the physical state of the patients， surgery， radiotherapy and chemotherapy are used in different combinations to fight against and control cancer. However， these commonly used methods may have side effects or no positive effect in terminal cancers， so new cancer treatments are urgently needed［4-5］.

In the last two decades， remarkable advances in nanotechnology and nanoscience have offered possibilities for overcoming the shortcomings in conventional cancer treatment methods［6］. Nanotechnology， a technique for studying and manipulating very small substances with nanoparticle sizes between 1-100 nm， has played an important role in developing new cancer treatment strategies［4， 7］. Several studies have demonstrated that nanoparticles have a certain efficacy in existing drug treatments. The combination of chemotherapeutic drugs with nanoparticles can increase the accumulation of drugs in cancerous tissues， enhance their penetration through cell membranes［8］， and improve the permeation and retention effect （EPR）， i.e.， the selective accumulation of nanoparticles or polymeric drugs in solid tumors. Due to the EPR effect， nanotechnology has been identified as a suitable platform for antitumor drug delivery［8］.

This review introduces the latest progress in synthesis and surface modifications of superparamagnetic iron oxide nanoparticles （SPIONs）， as well as its application in cancers.

Synthesis of SPIONs

Co-precipitation

Co-precipitation is the most common method used for the synthesis of SPIONs， in which iron precursors are reduced to iron oxides by mild reducing agents. Generally， SPIONs， Fe3O4 or γ-Fe2O3 are usually prepared in Fe2+ and Fe3+ solutions， through the addition of alkaline solution （e.g.， NaOH or NH4OH） as a precipitant［9］， in a non-oxidizing environment and at room temperature or high temperatures. The reaction equation is Fe2++2Fe3++8OH→Fe3O4+4H2O. The size， shape and composition of SPIONs depend greatly on salts （e.g.， chloride， sulfate and nitrate）， the ratio of Fe2+ to Fe3+ （r=Fe2+/Fe3+）， reaction temperature， pH and ionic strength of the medium［10， 11］. In the study of Jolivet et al.［12］， iron oxide nanoparticles （2-15 nm） were prepared via careful control of the ionic strength and pH of the solution. But the iron precursors in this method are susceptible to oxidation and reduction， resulting in unstable and inhomogeneous Fe3O4 NPs. To synthesize homogeneous iron oxide nanoparticles with ultra-small size， co-precipitation was conducted at a high temperature， using trithiol-terminated poly（methacrylic acid） （PMAA-PTTM） as stabilizer， and as a result， water soluble and biocompatible iron oxide nanoparticles with size 4.5±0.5 nm was obtained［13］. Iron oxide nanoparticles （3.3±0.5 nm） with smaller size than above nanoparticles were synthesized using the same method， but dissolving iron precursors in concentrated hydrogen chloride before the addition of precipitating agent.

Thermal decomposition

Thermal decomposition is one of the most widely used methods to produce highly crystalline and monodisperse SPIONs. This method has been used to precisely control the particle size and shape of SPIONs in organic solvents with high boiling points and at 200 ℃ or higher temperatures. For example， the ultra-small superparamagnetic iron oxide nanoparticles （USIONPs） （γ-Fe2O3）， less than 3 nm in size， synthesized using esters （diethylenglycolactetylacetonate and diethylenglycolpropionate） USIONPs as the solvent or coating stabilizer via thermal decomposition， are homogeneously dispersed in organic fluids［14］. In another study， USIONPs are synthesized via the thermal decomposition of iron-oleate complex， and USIONPs of different sizes （1.5-3.7 nm） were produced by changing the reaction temperature and the ratio of oleyl alcohol to oleic acid［15］.

Microemulsion

Microemulsions are thermodynamically stable dispersions of two mutually immiscible liquids （usually oil and water）［16］. With the aid of surfactants， mutually immiscible liquids can maintain a hydrophilic-lipophilic equilibrium in a single-phase solution［17］. The two most common microemulsions used for the synthesis of nanoparticles are water-in-oil （W/O） and oil-in-water （O/W）， respectively. According to Lopez-quintela et al.［18］， the synthesis of nanoparticles in microemulsions involves the following stages： （1） mixing of microemulsions， （2） exchange of reactants between nanodroplets， （3） nucleation， and （4） growth reaction. Hu et al.［19］ developed a novel method to produce iron oxide nanoparticles in situ in O/W microemulsions， which can improve the oil recovery efficiency. In the study of Lopez et al.［20］， a reverse microemulsion method was applied to control the size of iron oxide nanoparticles， which form droplets in organic solvents.

Hydrothermal synthesis

Hydrothermal synthesis is one of the most common methods used to prepare nanomaterials， by which many types of nanomaterials have been successfully synthesized. Nanomaterials can be produced by this approach at a wide range of temperatures from room temperature to very high temperatures. In addition， a high or low pressure can be chosen depending on the vapor pressure of the starting material in the reaction to control the morphology of the nanomaterials to be created. Hydrothermal synthesis has significant advantages over other methods， as nanomaterials with high vapor pressure can be produced by this way with minimum loss in materials. Moreover， the composition of nanomaterials can be controlled by liquid-phase or multiphase chemical reactions［21］. In the study of Da silva et al.［22］， magneto-luminescent nanocomposites were obtained by coating coprecipitated iron oxide nanoparticles with a functionalized carbon layer by hydrothermal treatment， using different organic precursors， D-glucose and p-phenylenediamine. The carbon layer played an important role in the structural， magnetic， and surface optical properties of the nanomaterials， and the D-glucose increased the saturation magnetization of the nanoparticles. Instead， p-phenylenediamine decreased the saturation magnetization of the nanoparticles. Also， nanoparticles with multicolored emissions on long-wavelength were found due to the carbon shell. So， nanomaterial properties can be tuned using different precursor reagents.

Surface Modification of SIONPs

SIONPs tend to aggregate into large clusters or be oxidized rapidly in tumor physiological environments due to their large surface area， high chemical reaction activity and surface properties， and thus lose their magnetic properties［23-25］. Therefore， surface modification of iron oxide nanoparticles is needed to improve the dispersion， surface activity of nanoparticles， physicochemical and mechanical properties and biocompatibility of nanoparticles［24-26］.

Surface coating with inorganic materials

Silica

Silica is the most common material used for nanoparticle surface modification. Silica nanoparticles promote the widespread use of novel multifunctional materials in biotechnology. Especially， the latest breakthroughs in the biomedical application of silica nanoparticles may herald the possibility of realizing individualized treatment. Silica nanoparticles are unique nanoparticles that combine the chemical and physical stability of silica with the pore-network structure of mesoporous materials， and have high loading capacity， controllable size and shape， good biocompatibility， and low cytotoxicity， which make them promising candidates for application in biotechnology［27-29］. Fathy et al.［30］ evaluated the effect of silica-coated SIONPs as a radiosensitizer and compared their therapeutic effect with that of iron oxide magnetic nanoparticles， and the results proved that SIONPs has a potential for improving the radiosensitivity of breast cancer. In the study of Sadegha et al.［31］， mesoporous silica coated SPIONs （mSiO2@SPIONs） containing curcumin （CUR） and silymarin （SIL） were prepared and evaluated on breast cancer cell line MCF-7， and the MTT results showed that mSiO2@SPIONs containing CUR+SIL reduced the IC50 in MCF-7 by approximately 50%， in comparison with that of the free drug mixture.

Carbon

Carbon-based inorganic materials have also been used for surface coating of SPIONs to improve their stability， biocompatibility and dispersion. In recent years， graphene has sparked enormous scientific interests worldwide due to its distinctive physicochemical properties such as large surface area， good electrical and thermal conductivity［24， 32］. In the study of Yousefi et al.［33］， the anticancer property of a novel ZnO/CNT@Fe3O4 nanocomposite in acute myelogenous leukemia （AML）-derived KG1 cells was evaluated， and the results showed that ZnO/CNT@Fe3O4 decreased the viability and metabolic activity of KG1 cells through induction of G1 arrest by increasing the expression of p21 and p27 cyclin-dependent kinase inhibitors and decreasing c-Myc transcription. In addition， the synergistic experiments showed that ZnO/CNT@Fe3O4 enhanced the toxic effects of vincristine on KG1 cells.

Metals

In recent decades， silver nanoparticles （AgNPs） have gained considerable attention in the medical and pharmaceutical fields due to their remarkable therapeutic properties. Studies have shown that AgNPs possess antivirus/anti-cancer activities and minimum toxicity［34-36］. Hosseini et al.［37］ reported that AgNPs have no harmful effects on humans and only kill viruses， bacteria and other eukaryotic microorganisms. Moreover， due to the high toxicity of other magnetic metal nanoparticles such as nickel and cobalt， FeONPs are known as the only magnetic nanoparticles approved by the US Food and Drug Administration （FDA） for clinical application［38-39］. Compared to other magnetic nanoparticles， FeONPs have negligible toxicity， good biocompatibility and biodegradability［40］， presenting promising potential in the treatment of tumors and iron-deficiency anemia［41］.

Surface coating with organometallics

Polyethylene glycol （PEG） is a commonly used water-soluble polymer， and PEG-coated iron oxide nanoparticles have been extensively reported and used in biomedical field［42］. In the study of Liu et al.［43］， Fe3O4/PEG 2 000/Cu nanocomposite was synthesized using PEG as stabilizer agent of CuNPs， and the experimental results showed that Fe3O4/PEG 2 000/Cu nanocomposite has high antioxidant activity， and the viability of malignant gastric cell lines NCI-N87 and MKN45 reduced dose-dependently in the presence of Fe3O4/PEG 2 000/Cu nanocomposite. Karaagac et al.［44］ produced PEG-coated iron oxide nanoparticles by a two-step synthesis route： coprecipitation and coating， and found that PEG coated iron oxide nanoparticles had higher saturation magnetization value than the uncoated iron oxide nanoparticles.

Qi NIE et al. Recent Progress in Superparamagnetic Iron Oxide Nanoparticles

Surface Modifications for Targeted Drug Delivery

Transferrin

Transferrin （Tf）， an important tumor-targeting ligand as well as a plasma glycoprotein that binds iron ions， is involved in the transport of ions and has low immunogenicity［45-46］. Tf has a high affinity for their receptors （TfR） overexpressed by a variety of tumor cells， especially those by breast cancer cells［46-47］. In phase I/II clinical trials， TfR overexpression in tumor tissues has been successfully constructed in targeted Tf-coupled drug delivery systems in cancer therapy. In the research of Ag Seleci et al.［48］， Tf-decorated niosomes with integrated magnetic iron oxide nanoparticles （MIONs） and quantum dots （QDs） were formulated （PEGNIO/QDs/MIONs/Tf） for efficient imaging of glioma， supported by magnetic and active targeting. And the results confirmed that Tf modification increased the antiproliferative activity of glioblastoma （GBM） cells， and the external magnetic field significantly enhanced cellular uptake of the niosomal formulation by GBM cells， which demonstrates that Tf modification has a great potential in dual targeting and imaging of gliomas［48］.

Folic acid

Folic acid is essential for the synthesis， modification and methylation of DNA and RNA， can promote cell growth， maintain normal cellular function， and prevent cancer［49］. It can retain its ability to bind to its receptors after attachment to nanomaterials and drugs， for the formation of folate-targeted drug delivery system， which can enter into the cells that express its receptors via receptor-mediated endocytosis［46］. Therefore， folic acid is widely used ligand， in combination with nanomaterials and/or antitumor drugs （e.g.， adriamycin and paclitaxel）， for cancer therapy and tumor imaging［46］. In the study of Ramezani et al.［50］， FA@HPG@Fe3O4 NPs were prepared by coating iron oxide nanoparticles with hyperbranched polyglycerol （HPG） and folic acid （FA）， and experimental results showed that compared with non-functionalized nanoparticles， the functionalized nanoparticles （10-20 nm） were less likely to aggregate， and able to increase cellular uptake and the t2-weighted signal intensity during MRI， demonstrating that FA@HPG@Fe3O4 nanoparticles have potential as anticancer drug delivery systems.

Antibodies

Antibodies， otherwise known as immunoglobulins， are glycoproteins of the immunoglobulin superfamily， and the main components of plasma gamma globulins. In most nanoparticular anti-tumor targeting research， antibodies are used to target disease-associated surface markers on cells. These markers （usually receptors， such as EGFR， VEGFR） are typically elevated or expressed in particular tumor-associated cells， and can be targeted to deliver chemotherapeutic drugs［51］.

EGFR

Epidermal growth factor receptor （EGFR） is a receptor tyrosine kinase belonging to the ErbB family of proteins. Ligand binding is required to activate the tyrosine kinase domain， and then activates the signaling pathways responsible for cell proliferation， angiogenesis migration and adhesion. These pathways are essential for the survival of cancer cells［52］. Previous studies on EGFR-targeted cancer therapy have shown that metal nanoparticles such as gold nanoparticles （AuNPs） are capable of enhancing the therapeutic efficacy and delaying the emergence of drug resistance. Besides， AuNPs as drug delivery agents can increase the pharmacokinetics of EGFR drugs， achieving high doses of targeted drug delivery［53］. Due to their large surface area for drug attachment， AuNPs can be an effective carrier for EGFR antibodies and tyrosine kinase inhibitors in molecular targeted cancer therapy［54］. Freis et al.［55］ coated EGFR-specific targeting ligands on the surface of iron oxide nanoparticles to prepare nanoparticles that are able to specifically recognize EGFR-positive FaDu and 93-Vu head and neck cancer cells. Compared to the bare iron oxide nanoparticles， the surface-coated iron oxide nanoparticles are able to enhance cellular internalization and thus increase drug accumulation in cancer tissues.

VEGF

Vascular endothelial growth factor （VEGF）， an important signaling protein that induces angiogenesis， can drive the migration of proliferation of vascular endothelial cells［56］. Therefore， anti-VEGF monoclonal antibodies such as bevacizumab， aflibercept， and anti-vegfr2 have been used for drug delivery to control the malignant development of vascular diseases via the inhibition of VEGF expression［46］. In the study of WANG et al.［57］， ECM bioscaffolds were derived from decellularized sheets and then modified with vascular endothelial growth factor （VEGF）-conjugated superparamagnetic iron oxide nanoparticles （Fe3O4 NP-VEGF）， and the results showed that the scaffolds can promote vascular endothelial cell generation and thus improve the regeneration of uroepithelial and smooth muscle cells［57］.

Applications of SIONPs

Applications of SIONPs as contrast agents

To improve the diagnostic accuracy of magnetic resonance imaging （MRI） using contrast agents has been an important issue in recent years. SIONPs are expected to be the next generation of contrast agents because of their excellent MRI performance， such as long circulation time after being appropriately surface-modified， high renal clearance and biological safety［58］. In the study of Kader et al.［59］， the SIONPs probe Feraheme was adopted as a contrast agent for visualizing prostate cancer treatment， leading to a large decrease in T1， T2， and T2* relaxation time. And the results also showed that macrophages in smaller tumors take up more iron than in larger tumors. This non-invasive method could help to detect tumors and to identify molecular characteristics.   In the study of Xia et al.［60］， LA-PEG-SPIONs were obtained by modifying SPIONs with lactobionic acid （LA） and PEG， and electron microscopy showed that LA-PEG-SPIONs were uniformly spherical， with regular morphology and good dispersion. Besides， LA-PEG-SPIONs had no toxicity or low toxicity to HepG2 cells and HeLa cells， even at 400 μg/mL. In vitro， MRI showed that the T2-weighted signal intensity of HepG2 cells was lower than that of HeLa cells. LA-PEG-SPION can specifically target liver cancer cells and may serve as a potential T2-weighted negative contrast in MRI. Yang et al.［61］ developed MRI nanoprobe Fe3O4-Met-Cy5.5 via conjugating notable hypoxia-sensitive metronidazole （Met） and Cy5.5 dye with ultrasmall iron oxide （Fe3O4） nanoparticles， and in vitro and in vivo experiments proved Fe3O4-Met-Cy5.5 has excellent performance in relaxivity， biocompatibility and hypoxia specificity， suggesting that it is highly feasible to use the probe as a T1-weighted MRI contrast agent to detect hypoxic area in tumors.

Application of SIONPs as drug carriers in tumor therapy

Glioblastoma （GBM）

GBM is the most common primary malignant central nervous system tumor. Regardless of multidisciplinary treatment， including maximal surgical resection， chemotherapy and radiotherapy， the survival for most patients is only 12-14 months［62］. Besides， the difficulty in GBM treatment is exacerbated by the drug resistance developed during chemotherapy. But SiRNAs can silence the genes that are involved in the drug resistance， thereby sensitizing tumor cells to drugs. In the study of Chung et al.［63］， iron oxide nanoparticles functionalized with peptides （NP-CTX-R10） were used to deliver siRNA to silence O6-methylguanine-DNA methyltransferase （MGMT） to sensitize tumor cells to alkylating drug， Temozolomide （TMZ）. And the results indicated that NP-siRNA is able to achieve up to 90% gene silencing. This nanoparticle formulation has the ability to protect siRNA from degradation and to efficiently deliver the siRNA to induce therapeutic gene knockdown.

Materials Institute Lavoisier （MILs）， a subclass of metal-organic frameworks （MOF） composed of trivalent transition metals and carboxylate ligands， has a promising potential in biomedical field due to its high biocompatibility and molecule loading ability［64］. In the study of Pulvirent et al.［65］， a hybrid system of MNPs@MIL with particle size ≤ 50 nm was prepared， which retains both the nanometer dimensions and the magnetic properties of the Fe3O4 nanoparticles and possesses increased the loading capability due to the highly porous Fe-MIL. In addition， it is able to load， carry and release more temozolomide （TMZ） for the treatment of GBM. The internalization of the MIL-modified system is more evident than bare MNPs at all the used concentrations both in the cytoplasm and in the nucleus， suggesting that it can be capable of overcoming the blood-brain barrier and targeting brain tumors.

Lung cancer

As one of the most common lung cancers， non-small cell lung cancer （NSCLC） accounts for about 85% of all long cancer cases［66］. NSCLC diagnosis and treatment with SIONPs have been extensively reported. For example， Ngema et al.［67］ developed CLA-coated PTX-SPIONs to enhance the drug loading of SPIONs for potential targeted therapy of NSCLC. The superparamagnetism of the CLA-coated PTX-SPIONs was confirmed， with saturation magnetization of 60 emu/g and 29 Oe coercivity. CLA-coated PTX-SPIONs had a drug loading efficiency of 98.5% and demonstrated sustained site-specific in vitro release of PTX over 24 h （i.e.， 94% at pH 6.8 mimicking the tumor microenvironment）. Enhanced anti-proliferative activity was also observed with the CLA-coated PTX-SPIONs against a lung adenocarcinoma （A549） cell line after 72 h， with a recorded cell viability of 17.1%. The CLA-coated PTX-SPIONs showed enhanced suppression on A549 cell proliferation compared to pristine PTX.

In the study Reczynska et al.［68］， in order to prevent unwanted release of irons， SPIONs were coated with silica layers of different morphologies： non-porous （@SiO2）， mesoporous （@mSiO2） or with a combination of non-porous and mesoporous layers （@SiO2@mSiO2） deposited via a sol-gel method. The experimental results proved that compared with the uncoated SPIONs， all the three types of silica coating significantly decreased iron release， and the Brunauer-Emmett-Teller （BET） surface area increased from （7.54±0.02） m2/g for SPIONs to （101.3±2.8） m2/g for SPION@mSiO2. In vitro experiment showed that SPION@mSiO2 can effectively inhibit the proliferation of malignant lung epithelial cell A549.

Breast cancer

Breast cancer is the most frequent malignant tumor in women. According to the International Agency for Research on Cancer in 2020， breast cancer accounted for 11.7% of all cancers all over the world， and approximately 2.26 million new cases of breast cancer are diagnosed every year［69］. The development of nanotechnology has brought new therapeutic agents and options for the treatment of breast cancer.

In the study of Candido et al.［70］， SPIONs were synthesized by the chemical coprecipitation method， stabilized by sodium citrate， and gold-coated. Then， SPIONs@Au were functionalized with EGF-α-lipoic acid and chlorin e6 （Ce6）-cysteamine complexes， composing a theranostic nanoprobe （TP）. The TPs uptake by the triple-negative breast cancer cell line MDA-MB-468 showed a continuous increase up to 240 min， demonstrating the presence and effectiveness of internalization of TPs in the cytoplasm and cell nuclei. PTT treatment showed the percentages of apoptotic breast cancer cells increased dramatically.

Osteoclasts and tumor cells interact synergistically to promote cancer progression inside the metastatic environment. Furin plays a key role in tumor cell invasion and bone resorptive function of osteoclasts. To investigate therapeutic strategies for breast cancer bone metastasis， PANG Y et al. developed bone targeting-Furin inhibition SPIO （BT-FI@SPIO） nanoparticles. In vitro and in vivo experiments showed that this system can effectively inhibit osteoclast bone resorption and breast cancer cell MDA-MB-231 invasion， and can alleviate osteolysis by reducing osteoclast-mediated bone destruction［71］.

Summary

In recent years， nanoparticles have been proven to be superior to conventional anticancer therapy in many ways， due to their outstanding superparamagnetic property， high biological activity， easy accumulation in cancer cells and low toxicity to humans. This paper introduces the latest progress in the synthesis and surface modification of nanoparticles， as well as their application as contrast agents and in cancers. These new technological approaches provide a solid theoretical basis for the innovation of nanotechnology and the development of cancer therapeutics.

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