3.4.2. Design of SPIONs Suitable for Hyperthermia and Immune System Targeting

SPIONs can be produced through physical, biological, or chemical routes. Nevertheless, chemical syntheses remain the main way to obtain SPIONs. The co-precipitation method has been the starting point for other approaches such as thermal decomposition methods, hydrothermal methods, solvothermal methods, sol-gel methods, micelle methods, and many other methods [97–99].

The method of synthesis drives the size, shape, colloidal stability, and magnetic properties of the SPIONs. For biomedical applications, SPIONs need to be modified to enhance their stability. This goal can be achieved through the grafting of various polymers such as PEG, polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), dextran, chitosan, and many others [97].

For instance, polymers such as PEG are now well-known to increase the biocompatibility, colloidal dispersion, and stability of SPIONs while conferring them relative stealthiness towards the RES. [100]. Silanes are also common coating polymers of SPIONs, used for example to modify the surface (aminosilane type shell) of NanoTherm® (size about 15 nm). This is the only SPION approved to treat glioblastoma with MHT induced with AMF [97]. Nevertheless, it is important to underline that NanoTherm® has to be injected directly into the tumor.

Many interesting preclinical/clinical studies on magnetic hyperthermia (MHT) with SPIONs have been carried out, but very few have led to advanced clinical phases [101]. In spite of its well-known efficacy on cancer cells, the main drawback of hyperthermia is its lack of selectivity between healthy and tumor tissues. To overcome this issue, SPI-ONs might be one of the most promising solutions if tumor targeting techniques are used (i.e., intra-tumor implantation, pharmacological targeting, and/or magnetic field application). In this context, several clinical trials have been performed with SPIONs, especially in the field of prostate cancer and glioblastoma [102]. Even if most studies have proven good efficacy, methodology/instrumentation issues impair the broader use of magnetic hyperthermia. Nevertheless, the case of Nanotherm® is particularly interesting and gives

rise to much hope in this field. Indeed, it is the only approved nanoparticle for MHT of glioblastoma (CE marking, approval in 2010 as a class III medical device) and has recently (2021) achieved a new accomplishment in prostate cancer, being allowed by the FDA to move towards a pivotal phase 2b clinical trial.

So, we could consider that the year 2021 is likely to be a turning point for MHT with the arrival in early clinical phases of new iron nanoparticles from the NoCanTher project (RCL-01, a 149 nm iron oxide nanoparticles coated with dextran) to treat locally advanced pancreatic ductal adenocarcinoma [103]. Based on their designs, we can consider that these products developed and used in clinical settings belong to the first generation of SPIONs. Indeed, these SPIONs are based on a magnetic core decorated with an organic coating without any targeting moieties. This justifies their implantation in situ with surgical procedures to achieve MHT with ad hoc devices. Moreover, the cancers addressed by these new therapies are well-known to be particularly challenging from a pharmacokinetics point of view (blood-brain barrier, blood-prostate barrier), justifying once again, the intratumor injection. We could consider that the intratumoral delivery of SPIONs for MHT is the counterpart of what is classically done in the framework of brachytherapy to treat many cancers (gynecological, prostate, and skin).

Based on these recent data, we assume that MHT with SPIONs is still in its infancy, paving the way to future smarter approaches. Thus, the modularity and theranostic capabilities of SPIONs should make it possible to design and develop a new generation of tumor-selective drugs until clinical phases. Ideally, this new generation should be suitable for the intravenous route with an optimal tumor uptake guided with MRI, allowing us to perform a safe and efficient MHT procedure.

So, for reasons of therapeutic refinement, SPIONs may also acquire active targeting capabilities through, for example, surface modification with antibodies, targeting peptides, or any other molecules with biological targeting capability [98]. However, it should be remembered that the changes in the surface of SPIONs may modulate the thickness of the overall surface coating, affecting the performances of T2 relaxation (MRI) and MHT [104,105]. Overall, when designing SPIONs for MHT, a balance must be achieved between the size of the magnetic core to maximize heat release (>10 nm) and colloidal stability in biological media required for intravenous injection (ideally <50 nm) [106]. Shape is also a major parameter to take into account when designing SPIONS for MHT purposes. For example, cubic-shaped SPIONs (from 17 to 61 nm) have been found to be more efficient in vitro to induce MHT when compared to spherical ones [107]. This effect was verified in vivo in subcutaneous A431 tumor-bearing mice, showing that cubic-shaped SPIONs coated with a polymer shell were able to induce effective MHT and heat-mediated chemotherapy [108].

Immune cells involved in the immunotherapy mechanism can be targeted with SPI-ONs. Thus, with a more or less sophisticated design based on the strategies previously evoked, SPIONs can be used for cancer vaccines, the guidance of magnetized cytotoxic cells to tumor sites, drug delivery of immune checkpoint inhibitors, the polarization of macrophages, and to trigger magnetic hyperthermia [109]. Of course, the modular construction of SPIONs and their magnetic properties allow us to consider combinatorial immunotherapies in the same nanomedicine [110]. Below, we will emphasize these approaches with recent studies given as examples of SPIONs designed for immunotherapeutic uses. For cancer vaccines, strategies based on ovalbumin bound to SPIONs (size around 200 nm, zeta potential around −22 mV) have been successfully evaluated as a vaccine delivery platform and immune potentiator, showing the activation of immune cells and cytokine production [111]. SPIONs can also be used as platforms to magnetically guide immune cells such as T cells to a region of interest. To do so, Ortega et al. designed several SPIONs coated with dimercaptosuccinic acid (DMSA), 3-aminopropyl-triethoxysilane (APS), or dextran (6 kDa). The size of these SPIONs ranged from 82 to 120 nm (zeta potential from −34 to +38 mV) and made it possible to activate in vivo the migration of T cells, loaded with SPIONs, through the application of an external magnetic field [112].

Another major way to target immunity with SPIONs is to target immune checkpoints since they are becoming a standard regimen in oncology. Very recently, Kiani et al. designed sophisticated SPIONs (90 nm, zeta potential of 28.6 mV), covered by chitosan, functionalized with TAT peptide (cell-penetrating peptide) and loaded with siRNA to silence two of the most important T-cell immune checkpoints (PD-1 and A2aR) [113]. These SPIONs significantly inhibited tumor growth (in CT26 and 4T1 mouse tumors) associated with an important anti-tumor immune response and survival time. SPIONs can also be designed to induce the repolarization of M2 to M1 (*vide infra*). In this way, Zhang et al. perform a study with differently charged SPIONs in order to see potential preferential differences in polarizing macrophages [114]. They synthesized three differently charged SPIONS (zeta potentials of +44.72 mV, −0.282 mV, and −27.31 mV for sizes about 19.4 nm, 15.9 nm, and 21.3 nm, respectively). Interestingly, they demonstrated that positively charged SPIONs had the highest cellular uptake and higher macrophage polarization effect (i.e., M2-like macrophages toward M1-like macrophages).

The shape of SPIONs is also an important parameter affecting the immunological response. Among the various existing shapes (e.g., spheres, rods, cubes, etc.) that have been designed so far, octapod- and plate-shaped SPIONs showed a higher immunomodulatory potential. The shape also influences the targeting and uptake within immune cells. For example, the internalization of spherical SPIONs is increased when compared to nonspherical ones. Conversely, at similar size and charge, spherical SPIONs are less efficient at diffusing across the vascular wall when compared to rod- or bar-shaped SPIONs [110].

In the context of immunotherapy, SPIONs are particularly suitable platforms for theranostic combinations. In this way, Wang et al. designed spherical SPIONs suitable for MRI, targeting M2-like macrophages and MHT in breast tumor-bearing mice [115]. They obtained a multifunctional SPION (hydrodynamic diameter of 20 nm), with efficient targeting capability, high relaxivity (149 s−1mM−<sup>1</sup> ), and satisfactory magnetic hyperthermia performance in vitro. In vivo MRI showed that M2-targeting SPIONs had a good biodistribution within tumors, also indicating the optimal timing for MHT. The MHT procedure induced both a decrease in the population of M2-like TAMs and tumoral volume associated with iTME remodeling (notably through a significant increase in CTLs). To go further, we invite the reader to consult a recent review related to the enhancement of CD8+ T-Cell-Mediated tumor immunotherapy via MHT used alone or in combination [116].

Due to the intrinsic versatility of nanomedicine, the various data in the literature show that there is no real consensus on the design of SPIONs. This suggests that the design of a given nanoparticle must be thought of in terms of its future application allowing us to imagine the most suitable specifications resulting from an optimal design. Figure 2 summarizes the design process of theranostic SPIONs emphasizing MHT and targeting M2-like tumor-associated macrophages. The first of these steps (Figure 2A) is the synthesis of the magnetic core (bare SPIONs), which influences its magnetic properties. Unless there is a magnetic field, magnetization equals 0. The core radius usually ranges from 5 to 15 nm. Many synthesis methods are available and drive the convenience of manufacturing, the control of shape, size, composition, and the polydispersity index (i.e., estimation of the average uniformity of a nanoparticle solution) of SPIONs. The second step is the surface engineering of SPIONs (Figure 2B). SPIONs can be coated with various organic moieties for biocompatibility (e.g., PEG, chitosan), targeting (e.g., mAbs, peptides), and theranostic (e.g., radionuclides, chemotherapeutics) purposes. Targeting molecules (e.g., carbohydrates such as mannose to target M2-like CD206 receptors) can also be bound to the biocompatible moieties. The surface engineering will influence the hydrodynamic size (i.e., core with shell and water coat—typically between 20 and 150 nm), zeta potential (i.e., the electric charge on the surface of a given nanoparticle, crucial for colloidal stability, typical absolute value: |30| mV), cellular uptake, toxicity, and hydrophilicity. Size also influences the EPR effect (i.e., passive targeting of tumors, up to 100–150 nm). Finally, the theranostic capabilities of SPIONs are assessed (Figure 2C). Due to their intrinsic superparamagnetic properties, the application of a magnetic field makes it possible to perform MRI, MPI,

and MHT (with AMF) and concentrate SPIONs within tumors. Interestingly, decorated SPIONs can target tumors and their microenvironment (e.g., M2-like macrophages through their CD206 receptor) to either exert their diagnostic (MRI, multimodal imaging such as PET-MRI, MPI) and/or their therapeutic (MHT, drug delivery) properties according to the design. In the context of immunotherapy, SPIONs might be particularly appealing through the combination in the same agent of immunogenic cell death inducers such as MHT and/or other thermal/phototherapies (e.g., photothermal therapy, photodynamic therapy), chemotherapy (e.g., doxorubicin), and radiotherapy in addition to macrophage repolarization from M2 to M1 phenotype. This combination makes it possible to boost both innate and adaptative immunity against tumors through the production of various tumoricidal mediators (cytokines such as IL1, TNF-α, and reactive oxygen species). Overall, in addition to these outstanding theranostic properties, SPIONs possess other many advantages such as long-term chemical stability, biocompatibility, and safety. Nevertheless, especially for MHT, the targeting strategies need to be improved to achieve a high concentration of SPIONs within targeted tissues to significantly reduce non-specific heating and increase efficacy. Moreover, in the context of clinical perspectives, all metallic material within 40 cm of the treatment area must be removed prior to alternating magnetic field exposure [117,118]. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 16 of 39

**Figure 2.** Design and theranostic applications of SPIONs suitable for MHT and macrophage targeting in cancers. Abbreviations. PEG: Poly-Ethylene Glycol; mAbs: monoclonal antibodies; EPR: Enhanced Permeability and Retention; MRI: Magnetic Resonance Imaging; MPI: Magnetic Particle Imaging; PET: Positron Emission Tomography; IL: Interleukin; TNF-α: Tumor Necrosis Factor-α; AMF: Alternating Magnetic Field; PFN: Perforins; GzmB: Granzyme B; IFNγ: Interferonγ. Created with BioRender.com. **Figure 2.** Design and theranostic applications of SPIONs suitable for MHT and macrophage targeting in cancers. Abbreviations. PEG: Poly-Ethylene Glycol; mAbs: monoclonal antibodies; EPR: Enhanced Permeability and Retention; MRI: Magnetic Resonance Imaging; MPI: Magnetic Particle Imaging; PET: Positron Emission Tomography; IL: Interleukin; TNF-α: Tumor Necrosis Factor-α; AMF: Alternating Magnetic Field; PFN: Perforins; GzmB: Granzyme B; IFNγ: Interferonγ. Created with BioRender.com.

#### **4. Targeting the Immune System with SPIONs 4. Targeting the Immune System with SPIONs**

#### *4.1. Magnetic Hyperthermia Based on SPIONs as an Immune Trigger against Tumors 4.1. Magnetic Hyperthermia Based on SPIONs as an Immune Trigger against Tumors*

Cancer cells are more sensitive to hyperthermia (elevation of temperature to 40–45 °C) than normal cells [119–121]. This may be because cancer cells have a more accelerated metabolism [122] or because there is poor vascular distribution in cancerous tissue, leading to an accumulation of fever and heat stress [123]. In this sense, several methods of increasing the temperature in order to eradicate tu-Cancer cells are more sensitive to hyperthermia (elevation of temperature to 40–45 ◦C) than normal cells [119–121]. This may be because cancer cells have a more accelerated metabolism [122] or because there is poor vascular distribution in cancerous tissue, leading to an accumulation of fever and heat stress [123].

mors have been investigated, such as those based on radiofrequency, microwaves, or ultrasound [124]. It is in this context that SPIONs can be used to generate heat via the use of electromagnetic energy, the so-called MHT [125]. Indeed, as previously seen and thanks to their magnetic properties, when subjected to an AMF, SPIONs are able to produce heat

that target cancer cells, it would then be possible to induce localized hyperthermia. This last point is particularly important since a key disadvantage of classical methods of hy-

Starting from this premise, only a few clinical trials have been conducted since 2006 to investigate the impact of thermotherapy based on SPIONs on different cancers, mostly glioblastoma and prostate cancer. SPION-based thermotherapy has also been investigated to treat other carcinomas (ovarian, cervical, and rectal) and sarcomas (chondro-, rhabomyo-, and parapharyngeal sarcoma) [126–129]. In general, these studies have shown that it was possible to have an increase in intratumoral temperature thanks to the combination of SPIONs and AMF. For instance, in prostate cancer, maximum temperatures up to 55 °C were reached [127]. Moreover, in glioblastoma, patients' overall survival was improved following MHT treatment [129]. In addition, both of these studies highlighted the fact that only moderate side effects were observed, with no serious complications [128,129].

perthermia induction is the lack of selectivity [118].

In this sense, several methods of increasing the temperature in order to eradicate tumors have been investigated, such as those based on radiofrequency, microwaves, or ultrasound [124]. It is in this context that SPIONs can be used to generate heat via the use of electromagnetic energy, the so-called MHT [125]. Indeed, as previously seen and thanks to their magnetic properties, when subjected to an AMF, SPIONs are able to produce heat [118]. Furthermore, since SPIONs can be functionalized on their surface with molecules that target cancer cells, it would then be possible to induce localized hyperthermia. This last point is particularly important since a key disadvantage of classical methods of hyperthermia induction is the lack of selectivity [118].

Starting from this premise, only a few clinical trials have been conducted since 2006 to investigate the impact of thermotherapy based on SPIONs on different cancers, mostly glioblastoma and prostate cancer. SPION-based thermotherapy has also been investigated to treat other carcinomas (ovarian, cervical, and rectal) and sarcomas (chondro-, rhabomyo-, and parapharyngeal sarcoma) [126–129]. In general, these studies have shown that it was possible to have an increase in intratumoral temperature thanks to the combination of SPIONs and AMF. For instance, in prostate cancer, maximum temperatures up to 55 ◦C were reached [127]. Moreover, in glioblastoma, patients' overall survival was improved following MHT treatment [129]. In addition, both of these studies highlighted the fact that only moderate side effects were observed, with no serious complications [128,129].

Recently, a phase 0 clinical trial (NCT02033447) investigating SPIONs-induced MHT with AMF has been completed but, as far as we know, no results have been published so far. Interestingly, another recent phase I (NCT04316091) clinical trial will study MHT in osteosarcoma with SPIONs triggered by spinning magnetic fields (SMF, a new type of magnetic field) in association with neoadjuvant chemotherapy [118]. Despite the fact that the feasibility of SPIONs-induced hyperthermia has been demonstrated at both preclinical and clinical levels, the low number of clinical trials can be partly explained by the fact that this thermotherapy is at the interface of several disciplines (physics, chemistry, biology, medicine, pharmacology) with potential issues to in designing ad hoc SPIONs. Therefore, a better understanding of the mechanism of this therapy in preclinical models, including its action on the immune system, is needed. Indeed, beyond the fact that hyperthermia can directly cause cancer cell death by necrotizing tissues [125], this therapy can also indirectly cause cancer cell death by activating antitumor immunity through ICD [124]. In this sense, Persano et al., in the context of glioblastoma, investigated the impact of magnetic hyperthermia on U87 cells in vitro following an iron oxide nanotube treatment. Interestingly, after thermotherapy, U87 cells displayed a different immunological profile (with an increase in stress-associated signals), making them more likely to be phagocyted by macrophages or killed by NK cells [130].

Other recent studies have demonstrated the impact of SPION-based MHT on the immune system. Carter et al. [125] demonstrated in a subcutaneous syngeneic (GL261 cells, glioblastoma) mouse model (C57BL/6), that magnetic hyperthermia treatment following intratumoral injection of Perimag-COOH SPIONs (dextran-coated, negatively charged and with a hydrodynamic diameter about 130 nm), induced an increase in the proportion of CD8+ T cells within tumors, which is a well-known good prognostic factor [131]. Carter et al. also demonstrated in this mouse model that magnetic hyperthermia treatment was able to reduce tumor growth when compared to control groups [125]. Covarrubias et al. showed in another syngeneic (4T1) mouse model (BALB/c), that IONPs-induced hyperthermia decreased immune cell subpopulations, including those from the innate system (such as neutrophils, dendritic cells, and macrophages) and adaptive system (i.e., CD4+ and CD8+ T cells). Interestingly, subsequent treatment with immune checkpoint inhibitors favored tumor repopulation with the infiltration of innate and adaptive immune cells within tumors [132]. More research is needed to fully assess the effects of SPION-based MHT on the tumor microenvironment. Finally, SPIONs may be useful in treating tumors, in addition to their capacity to cause hyperthermia, by reversing the immunosuppressive tumor microenvironment, which includes, among other things, their influence on macrophage polarization.
