Magnetic Nanoparticles: A Review on Synthesis, Characterization, Functionalization, and Biomedical Applications.

Nowadays, magnetic nanoparticles (MNPs) are applied in numerous fields, especially in biomedical applications. Since biofluidic samples and biological tissues are nonmagnetic, negligible background signals can interfere with the magnetic signals from MNPs in magnetic biosensing and imaging applications. In addition, the MNPs can be remotely controlled by magnetic fields, which make it possible for magnetic separation and targeted drug delivery. Furthermore, due to the unique dynamic magnetizations of MNPs when subjected to alternating magnetic fields, MNPs are also proposed as a key tool in cancer treatment, an example is magnetic hyperthermia therapy. Due to their distinct surface chemistry, good biocompatibility, and inducible magnetic moments, the material and morphological structure design of MNPs has attracted enormous interest from a variety of scientific domains. Herein, a thorough review of the chemical synthesis strategies of MNPs, the methodologies to modify the MNPs surface for better biocompatibility, the physicochemical characterization techniques for MNPs, as well as some representative applications of MNPs in disease diagnosis and treatment are provided. Further portions of the review go into the diagnostic and therapeutic uses of composite MNPs with core/shell structures as well as a deeper analysis of MNP properties to learn about potential biomedical applications.

Fe3O4SPIONs in cancer theranostics-structure versus interactions with proteins and methods of their investigation.

As the second leading cause of death worldwide, neoplastic diseases are one of the biggest challenges for public health care. Contemporary medicine seeks potential tools for fighting cancer within nanomedicine, as various nanomaterials can be used for both diagnostics and therapies. Among those of particular interest are superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties,. However, while the number of new SPIONs, suitably modified and functionalized, designed for medical purposes, has been gradually increasing, it has not yet been translated into the number of approved clinical solutions. The presented review covers various issues related to SPIONs of potential theranostic applications. It refers to structural considerations (the nanoparticle core, most often used modifications and functionalizations) and the ways of characterizing newly designed nanoparticles. The discussion about the phenomenon of protein corona formation leads to the conclusion that the scarcity of proper tools to investigate the interactions between SPIONs and human serum proteins is the reason for difficulties in introducing them into clinical applications. The review emphasizes the importance of understanding the mechanism behind the protein corona formation, as it has a crucial impact on the effectiveness of designed SPIONs in the physiological environment.

Magnetic Hyperthermia in Glioblastoma Multiforme Treatment.

Glioblastoma multiforme (GBM) represents one of the most critical oncological diseases in neurological practice, being considered highly aggressive with a dismal prognosis. At a worldwide level, new therapeutic methods are continuously being researched. Magnetic hyperthermia (MHT) has been investigated for more than 30 years as a solution used as a single therapy or combined with others for glioma tumor assessment in preclinical and clinical studies. It is based on magnetic nanoparticles (MNPs) that are injected into the tumor, and, under the effect of an external alternating magnetic field, they produce heat with temperatures higher than 42 °C, which determines cancer cell death. It is well known that iron oxide nanoparticles have received FDA approval for anemia treatment and to be used as contrast substances in the medical imagining domain. Today, energetic, efficient MNPs are developed that are especially dedicated to MHT treatments. In this review, the subject's importance will be emphasized by specifying the number of patients with cancer worldwide, presenting the main features of GBM, and detailing the physical theory accompanying the MHT treatment. Then, synthesis routes for thermally efficient MNP manufacturing, strategies adopted in practice for increasing MHT heat performance, and significant in vitro and in vivo studies are presented. This review paper also includes combined cancer therapies, the main reasons for using these approaches with MHT, and important clinical studies on human subjects found in the literature. This review ends by describing the most critical challenges associated with MHT and future perspectives. It is concluded that MHT can be successfully and regularly applied as a treatment for GBM if specific improvements are made.

In Vitro Superparamagnetic Hyperthermia Employing Magnetite Gamma-Cyclodextrin Nanobioconjugates for Human Squamous Skin Carcinoma Therapy.

In vitro alternative therapy of human epidermoid squamous carcinoma (A431) by superparamagnetic hyperthermia (SPMHT) using Fe3O4 (magnetite) superparamagnetic nanoparticles (SPIONs) with an average diameter of 15.8 nm, bioconjugated with hydroxypropyl gamma-cyclodextrins (HP-γ-CDs) by means of polyacrylic acid (PAA) biopolymer, is presented in this paper. The therapy was carried out at a temperature of 43 °C for 30 min using the concentrations of Fe3O4 ferrimagnetic nanoparticles from nanobioconjugates of 1, 5, and 10 mg/mL nanoparticles in cell suspension, which were previously found by us to be non-toxic for healthy cells (cell viabilities close to 100%), according to ISO standards (cell viability must be greater than 70%). The temperature for the in vitro therapy was obtained by the safe application (without exceeding the biological limit and cellular damage) of an alternating magnetic field with a frequency of 312.4 kHz and amplitudes of 168, 208, and 370 G, depending on the concentration of the magnetic nanoparticles. The optimal concentration of magnetic nanoparticles in suspension was found experimentally. The results obtained after the treatment show its high effectiveness in destroying the A431 tumor cells, up to 83%, with the possibility of increasing even more, which demonstrates the viability of the SPMHT method with Fe3O4-PAA-(HP-γ-CDs) nanobioconjugates for human squamous cancer therapy.

Tumor-Homing Peptides as Crucial Component of Magnetic-Based Delivery Systems: Recent Developments and Pharmacoeconomical Perspective.

According to data from the World Health Organization (WHO), cancer is considered to be one of the leading causes of death worldwide, and new therapeutic approaches, especially improved novel cancer treatment regimens, are in high demand. Considering that many chemotherapeutic drugs tend to have poor pharmacokinetic profiles, including rapid clearance and limited on-site accumulation, a combined approach with tumor-homing peptide (THP)-functionalized magnetic nanoparticles could lead to remarkable improvements. This is confirmed by an increasing number of papers in this field, showing that the on-target peptide functionalization of magnetic nanoparticles improves their penetration properties and ensures tumor-specific binding, which results in an increased clinical response. This review aims to highlight the potential applications of THPs in combination with magnetic carriers across various fields, including a pharmacoeconomic perspective.

Magnetite Nanoparticle Assemblies and Their Biological Applications: A Review.

Magnetite nanoparticles (Fe3O4 NPs) have garnered significant attention over the past twenty years, primarily due to their superparamagnetic properties. These properties allow the NPs to respond to external magnetic fields, making them particularly useful in various technological applications. One of the most fascinating aspects of Fe3O4 NPs is their ability to self-assemble into complex structures. Research over this period has focused heavily on how these nanoparticles can be organized into a variety of superstructures, classified by their dimensionality-namely one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) configurations. Despite a wealth of studies, the literature lacks a systematic review that synthesizes these findings. This review aims to fill that gap by providing a thorough overview of the recent progress made in the fabrication and organization of Fe3O4 NP assemblies via a bottom-up self-assembly approach. This methodology enables the controlled construction of assemblies at the nanoscale, which can lead to distinctive functionalities compared to their individual counterparts. Furthermore, the review explores the diverse applications stemming from these nanoparticle assemblies, particularly emphasizing their contributions to important areas such as imaging, drug delivery, and the diagnosis and treatment of cancer.

Influence of SPION Surface Coating on Magnetic Properties and Theranostic Profile.

This study aimed to develop multifunctional nanoplatforms for both cancer imaging and therapy using superparamagnetic iron oxide nanoparticles (SPIONs). Two distinct synthetic methods, reduction-precipitation (MR/P) and co-precipitation at controlled pH (MpH), were explored, including the assessment of the coating's influence, namely dextran and gold, on their magnetic properties. These SPIONs were further functionalized with gadolinium to act as dual T1/T2 contrast agents for magnetic resonance imaging (MRI). Parameters such as size, stability, morphology, and magnetic behavior were evaluated by a detailed characterization analysis. To assess their efficacy in imaging and therapy, relaxivity and hyperthermia experiments were performed, respectively. The results revealed that both synthetic methods lead to SPIONs with similar average size, 9 nm. Mössbauer spectroscopy indicated that samples obtained from MR/P consist of approximately 11-13% of Fe present in magnetite, while samples obtained from MpH have higher contents of 33-45%. Despite coating and functionalization, all samples exhibited superparamagnetic behavior at room temperature. Hyperthermia experiments showed increased SAR values with higher magnetic field intensity and frequency. Moreover, the relaxivity studies suggested potential dual T1/T2 contrast agent capabilities for the coated SPpH-Dx-Au-Gd sample, thus demonstrating its potential in cancer diagnosis.

Optimal heat transport induced by magnetic nanoparticle delivery in vascularised tumours.

We describe a novel mathematical model for blood flow, delivery of nanoparticles, and heat transport in vascularised tumour tissue. The model, which is derived via the asymptotic homogenisation technique, provides a link between the macroscale behaviour of the system and its underlying, tortuous micro-structure, as parametrised in Penta and Ambrosi (2015). It consists of a double Darcy's law, coupled with a double advection-diffusion-reaction system describing heat transport, and an advection-diffusion-reaction equation for transport and adhesion of particles. Particles are assumed sufficiently large and do not extravasate to the tumour interstitial space but blood and heat can be exchanged between the two compartments. Numerical simulations of the model are performed using a finite element method to investigate cancer hyperthermia induced by the application of magnetic field applied to injected iron oxide nanoparticles. Since tumour microvasculature is more tortuous than that of healthy tissue and thus suboptimal in terms of fluid and drug transport, we study the influence of the vessels' geometry on tumour temperature. Effective and safe hyperthermia treatment requires tumour temperature within certain target range, generally estimated between 42 °C and 46 °C, for a certain target duration, typically 0.5h to 2h. As temperature is difficult to measure in situ, we use our model to determine the ranges of tortuosity of the microvessels, magnetic intensity, injection time, wall shear stress rate, and concentration of nanoparticles required to achieve given target conditions.

Optimization of cobalt ferrite magnetic nanoparticle as a theranostic agent: MRI and hyperthermia.

OBJECTIVE: Magnetic nanoparticles (MNPs) are considered a theranostic agent in MR imaging, playing an effective role in inducing magnetic hyperthermia. Since, high-performance magnetic theranostic agents are characterized by superparamagnetic behavior and high anisotropy, in this study, cobalt ferrite MNPs were optimized and investigated as a theranostic agent. METHODS: CoFe2O4@Au@dextran particles were synthesized and characterized by DLS, HRTEM, SEM, XRD, FTIR, and VSM methods. After cytotoxicity evaluation, MR imaging parameters (r1, r2 and r2 / r1) were calculated for these nanostructures. Afterward, magnetic hyperthermia at the frequency of 425 kHz was applied to calculate specific loss power (SLP). RESULTS: Formation of CoFe2O4@Au@dextran was confirmed by UV-Visible spectrophotometry. On the basis of the relaxometric and hyperthermia induction findings of nanostructures in all stages of synthesis, the CoFe2O4@Au@dextran could produce the highest parameters of r2 and r2/r1 and SLP with values ​​of 389.7, 51.2 mM-1 s-1, and 2449 W/g, respectively. CONCLUSION: The formation of multi-core MNPs by dextran coating is expected to improve the magnetic properties of the nanostructure, leading to optimization of theranostic parameters, so that CoFe2O4@Au@dextran NPs can create contrast-enhanced images more than three times the clinical use and require less contrast agent, reducing side effects. Accordingly, CoFe2O4@Au@dextran can be introduced as a suitable theranostic nanostructure with optimal efficiency.

Improving the Efficacy of Magnetic Nanoparticle-Mediated Hyperthermia Using Trapezoidal Pulsed Electromagnetic Fields as an In Vitro Anticancer Treatment in Melanoma and Glioblastoma Multiforme Cell Lines.

Magnetic hyperthermia (MHT) is an oncological therapy that uses magnetic nanoparticles (MNPs) to generate localized heat under a low-frequency alternating magnetic field (AMF). Recently, trapezoidal pulsed alternating magnetic fields (TPAMFs) have proven their efficacy in enhancing the efficiency of heating in MHT as compared to the sinusoidal one. Our study aims to compare the TPAMF waveform's killing effect against the sinusoidal waveform in B16F10 and CT2A cell lines to determine more efficient waveforms in causing cell death. For that purpose, we used MNPs and different AMF waveforms: trapezoidal (TP), almost-square (TS), triangular (TR), and sinusoidal signal (SN). MNPs at 1 and 4 mg/mL did not affect cell viability during treatment. The exposition of B16F10 and CT2A cells to only AMF showed nonsignificant mortality. Hence, the synergetic effect of the AMF and MNPs causes the observed cell death. Among the explored cases, the nonharmonic signals demonstrated better efficacy than the SN one as an MHT treatment. This study has revealed that the application of TP, TS, or TR waveforms is more efficient and has considerable capability to increase cancer cell death compared to the traditional sinusoidal treatment. Overall, we can conclude that the application of nonharmonic signals enhances MHT treatment efficiency against tumor cells.

198Au-Coated Superparamagnetic Iron Oxide Nanoparticles for Dual Magnetic Hyperthermia and Radionuclide Therapy of Hepatocellular Carcinoma.

This study was performed to synthesize a radiopharmaceutical designed for multimodal hepatocellular carcinoma (HCC) treatment involving radionuclide therapy and magnetic hyperthermia. To achieve this goal, the superparamagnetic iron oxide (magnetite) nanoparticles (SPIONs) were covered with a layer of radioactive gold (198Au) creating core-shell nanoparticles (SPION@Au). The synthesized SPION@Au nanoparticles exhibited superparamagnetic properties with a saturation magnetization of 50 emu/g, which is lower than reported for uncoated SPIONs (83 emu/g). Nevertheless, the SPION@Au core-shell nanoparticles showed a sufficiently high saturation magnetization value which allows them to reach a temperature of 43 °C at a magnetic field frequency of 386 kHz. The cytotoxic effect of nonradioactive and radioactive SPION@Au-polyethylene glycol (PEG) bioconjugates was carried out by treating HepG2 cells with various concentrations (1.25-100.00 µg/mL) of the compound and radioactivity in range of 1.25-20 MBq/mL. The moderate cytotoxic effect of nonradioactive SPION@Au-PEG bioconjugates on HepG2 was observed. The cytotoxic effect associated with the ß- radiation emitted by 198Au was much greater and already reaches a cell survival fraction below 8% for 2.5 MBq/mL of radioactivity after 72 h. Thus, the killing of HepG2 cells in HCC therapy should be possible due to the combination of the heat-generating properties of the SPION-198Au-PEG conjugates and the radiotoxicity of the radiation emitted by 198Au.

Towards Regenerative Audiology: Immune Modulation of Adipose-Derived Mesenchymal Cells Preconditioned with Citric Acid-Coated Antioxidant-Functionalized Magnetic Nanoparticles.

Introduction and Background: Based on stem cells, bioactive molecules and supportive structures, regenerative medicine (RM) is promising for its potential impact on field of hearing loss by offering innovative solutions for hair cell rescue. Nanotechnology has recently been regarded as a powerful tool for accelerating the efficiency of RM therapeutic solutions. Adipose-derived mesenchymal cells (ADSCs) have already been tested in clinical trials for their regenerative and immunomodulatory potential in various medical fields; however, the advancement to bedside treatment has proven to be tedious. Innovative solutions are expected to circumvent regulatory and manufacturing issues related to living cell-based therapies. The objectives of the study were to test if human primary ADSCs preconditioned with magnetic nanoparticles coated with citric acid and functionalized with antioxidant protocatechuic acid (MNP-CA-PCA) retain their phenotypic features and if conditioned media elicit immune responses in vitro. MNP-CA-PCA was synthesized and characterized regarding size, colloidal stability as well as antioxidant release profile. Human primary ADSCs preconditioned with MNP-CA-PCA were tested for viability, surface marker expression and mesenchymal lineage differentiation potential. Conditioned media (CM) from ADSCs treated with MNP-CA-PCA were tested for Il-6 and IL-8 cytokine release using ELISA and inhibition of lectin-stimulated peripheral blood monocyte proliferation. Results: MNP-CA-PCA-preconditioned ADSCs display good viability and retain their specific mesenchymal stem cell phenotype. CM from ADSCs conditioned with MNP-CA-PCA do not display increased inflammatory cytokine release and do not induce proliferation of allergen-stimulated allogeneic peripheral blood monocytes in vitro. Conclusions: While further in vitro and in vivo tests are needed to validate these findings, the present results indicated that CM from ADSCs preconditioned with MNP-CA-PCA could be developed as possible cell-free therapies for rescuing auditory hair cells.

[Surface modification of multifunctional ferrite magnetic nanoparticles and progress in biomedicine].

Magnetic ferrite nanoparticles (MFNPs) have great application potential in biomedical fields such as magnetic resonance imaging, targeted drugs, magnetothermal therapy and gene delivery. MFNPs can migrate under the action of a magnetic field and target specific cells or tissues. However, to apply MFNPs to organisms, further modifications on the surface of MFNPs are required. In this paper, the common modification methods of MFNPs are reviewed, their applications in medical fields such as bioimaging, medical detection, and biotherapy are summarized, and the future application directions of MFNPs are further prospected.

A review on magnetic and spintronic neurostimulation: challenges and prospects.

In the treatment of neurodegenerative, sensory and cardiovascular diseases, electrical probes and arrays have shown quite a promising success rate. However, despite the outstanding clinical outcomes, their operation is significantly hindered by non-selective control of electric fields. A promising alternative is micromagnetic stimulation (µMS) due to the high permeability of magnetic field through biological tissues. The induced electric field from the time-varying magnetic field generated by magnetic neurostimulators is used to remotely stimulate neighboring neurons. Due to the spatial asymmetry of the induced electric field, high spatial selectivity of neurostimulation has been realized. Herein, some popular choices of magnetic neurostimulators such as microcoils (µcoils) and spintronic nanodevices are reviewed. The neurostimulator features such as power consumption and resolution (aiming at cellular level) are discussed. In addition, the chronic stability and biocompatibility of these implantable neurostimulator are commented in favor of further translation to clinical settings. Furthermore, magnetic nanoparticles (MNPs), as another invaluable neurostimulation material, has emerged in recent years. Thus, in this review we have also included MNPs as a remote neurostimulation solution that overcomes physical limitations of invasive implants. Overall, this review provides peers with the recent development of ultra-low power, cellular-level, spatially selective magnetic neurostimulators of dimensions within micro- to nano-range for treating chronic neurological disorders. At the end of this review, some potential applications of next generation neuro-devices have also been discussed.

90Y-CA/SPIONs for dual magnetic hyperthermia-radionuclide nanobrachytherapy of solid tumours.

Radiolabelled superparamagnetic iron oxide nanoparticles (SPIONs) are a promising nanomaterial for the development of dual radiation/hyperthermia cancer therapy. To that purpose, flower-shaped SPIONs with an exceptional heating capability were synthesised and coated with citrate, dextran or (3-aminopropyl)triethoxysilane. Both non-coated and coated SPIONs were nontoxic to CT-26 mouse colon cancer cells up to 1.0 mg ml-1in vitro. In an oscillating magnetic field, citrate-coated SPIONs (CA/SPIONs) displayed the highest heating rate (SAR âˆ¼ 253 W g-1) and the strongest hyperthermia effects against CT-26 cells. Labelling of the CA/SPIONs by the90Y radionuclide, emitting ß-radiation with an average/maximum energy of 0.94/2.23 MeV, and deep tissue penetration generated90Y-CA/SPIONs intended for the therapy of solid tumours. However, intravenous injection of90Y-CA/SPIONs in CT-26 xenograft-bearing mice resulted in low tumour accumulation. On the contrary, intratumoural injection resulted in long-term retention at the injection site. A single intratumoural injection of 0.25 mg CA/SPIONs followed by 30-min courses of magnetic hyperthermia for four consecutive days caused a moderate antitumour effect against CT-26 and 4T1 mouse tumour xenografts. Intratumoural application of 1.85 MBq/0.25 mg90Y-CA/SPIONs, alone or combined with hyperthermia, caused a significant (P ≤ 0.01) antitumour effect without signs of systemic toxicity. The results confirm the suitability of90Y-CA/SPIONs for monotherapy or dual magnetic hyperthermia-radionuclide nanobrachytherapy (NBT) of solid tumours.

Combination of selol nanocapsules and magnetic hyperthermia hinders breast tumor growth in aged mice after a short-time treatment.

Short time treatment with reduced dosages of selol-loaded PLGA nanocapsules (NcSel) combined with magnetic hyperthermia (MHT) is evaluated in aged Erhlich tumor-bearing mice. Clinical, hematological, biochemical, genotoxic and histopathological parameters are assessed during 7 d treatment with NcSel and MHT, separately or combined. The time evolution of the tumor volume is successfully modeled using the logistic mathematical model. The combined therapy comprising NcSel and MHT is able to hinder primary tumor growth and a case of complete tumor remission is recorded. Moreover, no metastasis was diagnosed and the adverse effects are negligible. NcSel plus MHT may represent an effective and safe alternative to cancer control in aged patients. Future clinical trials are encouraged.

Emerging trends in the nanomedicine applications of functionalized magnetic nanoparticles as novel therapies for acute and chronic diseases.

High-quality point-of-care is critical for timely decision of disease diagnosis and healthcare management. In this regard, biosensors have revolutionized the field of rapid testing and screening, however, are confounded by several technical challenges including material cost, half-life, stability, site-specific targeting, analytes specificity, and detection sensitivity that affect the overall diagnostic potential and therapeutic profile. Despite their advances in point-of-care testing, very few classical biosensors have proven effective and commercially viable in situations of healthcare emergency including the recent COVID-19 pandemic. To overcome these challenges functionalized magnetic nanoparticles (MNPs) have emerged as key players in advancing the biomedical and healthcare sector with promising applications during the ongoing healthcare crises. This critical review focus on understanding recent developments in theranostic applications of functionalized magnetic nanoparticles (MNPs). Given the profound global economic and health burden, we discuss the therapeutic impact of functionalized MNPs in acute and chronic diseases like small RNA therapeutics, vascular diseases, neurological disorders, and cancer, as well as for COVID-19 testing. Lastly, we culminate with a futuristic perspective on the scope of this field and provide an insight into the emerging opportunities whose impact is anticipated to disrupt the healthcare industry.

Simulation of heat transfer, mass transfer and tissue damage in magnetic nanoparticle hyperthermia with blood vessels.

Numerical simulation of magnetic nanoparticle hyperthermia for cancer treatment has been investigated in this study. The presented simulation did account for the effects of fluid flow, mass flow, and heat transfer during the MNP hyperthermia. The tumor was assumed to be a porous slab, 30% of which had been necrosed previously, with two capillaries, where magnetic nanoparticles were added into the bloodstream and distributed in the tumor by blood flow through capillaries. Fluid flow, mass transfer by capillaries, and interstitial tissues have been coupled in this study. Furthermore, tumor tissue damage has been calculated using a thermal damage indicator. The goal of this research is to find an optimum injection duration and exposure time in order to maximize hyperthermia treatment effectiveness using the BOBYQA optimization method. At the end of the 1-h time hyperthermia treatment, most of the non-necrotic tissue of the tumor were damaged. Moreover, the fraction of damaged tissue increased to more than 90% in some parts of the tumor. Results of this study indicate that MNP hyperthermia with the proposed setup can effectively damage the tumor in just one session, making it more susceptible to complementary therapies such as radiotherapy or chemotherapy.

Magnetic Hyperthermia Nanoarchitectonics via Iron Oxide Nanoparticles Stabilised by Oleic Acid: Anti-Tumour Efficiency and Safety Evaluation in Animals with Transplanted Carcinoma.

In this study, we developed iron oxide nanoparticles stabilised with oleic acid/sodium oleate that could exert therapeutic effects for curing tumours via magnetic hyperthermia. A suspension of iron oxide nanoparticles was produced and characterised. The toxicity of the synthesised composition was examined in vivo and found to be negligible. Histological examination showed a low local irritant effect and no effect on the morphology of the internal organs. The efficiency of magnetic hyperthermia for the treatment of transplanted Walker 256 carcinoma was evaluated. The tumour was infiltrated with the synthesised particles and then treated with an alternating magnetic field. The survival rate was 85% in the studied therapy group of seven animals, while in the control group (without treatment), all animals died. The physicochemical and pharmaceutical properties of the synthesised fluid and the therapeutic results, as seen in the in vivo experiments, provide insights into therapeutic hyperthermia using injected magnetite nanoparticles.

Immunomodulation of Melanoma by Chemo-Thermo-Immunotherapy Using Conjugates of Melanogenesis Substrate NPrCAP and Magnetite Nanoparticles: A Review.

A major advance in drug discovery and targeted therapy directed at cancer cells may be achieved by the exploitation and immunomodulation of their unique biological properties. This review summarizes our efforts to develop novel chemo-thermo-immunotherapy (CTI therapy) by conjugating a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine), with magnetite nanoparticles (MNP). In our approach, NPrCAP provides a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It also functions as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Moreover, the utilization of MNP is a platform to develop thermo-immunotherapy because of heat shock protein (HSP) expression upon heat generation in MNP by exposure to an alternating magnetic field (AMF). This comprehensive review covers experimental in vivo and in vitro mouse melanoma models and preliminary clinical trials with a limited number of advanced melanoma patients. We also discuss the future directions of CTI therapy.