Fe3O4 SPIONs 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.


Introduction
Neoplasm (neoplasma in Latin) is the term referring to a group of diseases caused by the uncontrollable growth and proliferation of cells in the human body.In the correctly functioning organism, cells proliferate and differentiate to meet the current demands and their apoptosis (programmed cell death) is caused by age or damage.As a result of certain mutations, either hereditary or caused by a range of external factors, human organism may lose control over these processes [1].There is a large variety of direct causes of genetic material changes leading to neoplastic cells' appearance.The most common ones include exposure to irradiation (Gamma, Roentgen, UV) and some chemical agents like asbestos compounds and aflatoxins, smoking, drinking alcohol or all kinds of infections caused by certain viruses (e.g.HPV).Other external factors that may stimulate the development of neoplastic disease are insufficient physical activity, unhealthy diet and obesity [2].It should also be mentioned that with age, Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. the effectiveness of the natural repair mechanisms in the organism decreases, increasing the risk of neoplastic disease appearance.
Treatment of neoplastic diseases is one of the greatest challenges of contemporary medicine.As reported in a recent study, even considering COVID-19-related death, neoplastic diseases are still the second most frequent cause of death [3,4].The coronavirus pandemic was the cause of difficulties in access to cancer diagnosis and treatment due to healthcare settings closures, disruptions in employment and health insurance, and fear of COVID-19 exposure.The consequences of this period are still visible and will lead to an increase in mortality.The number of cases and deaths can be estimated based on the data from previous years.However, the proper quantification will require a few more years due to the lag in the population-based cancer incidence and mortality data [5].Although the role of prophylactics in restricting the cancer incidence rate can hardly be overestimated, the development of diagnostic methods allowing early detection of neoplastic changes and therapies leading to the elimination of neoplastic cells is vital.The most often-used methods of cancer treatment include radiotherapy, chemotherapy, and direct surgical intervention.Unfortunately, despite considerable progress in the development of medical sciences, these methods show several of drawbacks.Surgical operations of neoplastic tumours are most effective at the early stages of cancer development, before metastasis, while radiotherapy and chemotherapy are charged with a number of side effects because of the lack of specificity [6,7].That is why intense research is carried out to design alternative effective therapies for neoplastic diseases or to improve the already known therapies (e.g. by using targeted drug delivery or bioimaging).
Nanomedicine may be a potential response to the challenges of contemporary medicine by providing new tools for diagnostics and therapies for neoplastic diseases [8].This interdisciplinary area, combining elements of medicine, chemistry, biology, and material engineering, is based on the use of nanomaterials, defined as structures with at least one dimension in the range smaller than 100 nm.Because of the size, nanoparticles' physicochemical properties differ from those of macroscale materials.The differences are mainly related to the high surface area to volume ratios of nanostructures, resulting in their unique optical, electric and magnetic properties [9,10].The possibility of biomedical use of nanomaterials is closely related to their size.To be able to apply them for medical purposes, nanoparticles must be greater than 10 nm to avoid removal through renal clearance and smaller than 180-200 nm to avoid the risk of being captured by the phagocytic system, leading to their accumulation in the spleen or the liver [11].Regardless of size, nanomaterials introduced into the bloodstream will undergo opsonization processes (the attachment of particles promoting phagocytosis, e.g.immunoglobulins).For this reason, while designing potential nanostructures for anticancer therapies, it is essential to understand the mechanisms behind the attachment of proteins to the surface of those structures.This research could help increase their circulation time in the human body and, thus, the effectiveness of the therapy.The most important types of medically attractive nanomaterials that may find application in anticancer therapies are presented in figure 1 [10,12].

Superparamagnetic iron oxide nanoparticles
Superparamagnetic iron oxide nanoparticles (SPIONs) represent the group of metallic nanoparticles with structures based on the crystals of α-Fe 2 O 3 (hematite), γ-Fe 2 O 3 (maghemite) or Fe 3 O 4 (magnetite) [9].These nanoparticles are composed primarily of iron oxides.However, the presence of other transition metal ions (such as Co, Ni, Zn, or Cu) used as dopants can significantly affect their properties, allowing, for example, for their use as catalysts in various organic-based reactions [13].They have enjoyed much interest because of their properties, including high stability, low toxicity, biocompatibility and superparamagnetism [14].What distinguishes SPIONs from other magnetic nanoparticles?Unlike ferromagnets, their magnetism can be externally controlled.Due to their size being smaller than the size of the single magnetic domain (below 30 nm), they exhibit magnetic properties only in the presence of an external magnetic field.This can be proven by conducting a hysteresis loop.The particle is subjected to a magnetic field of increasing strength until it reaches saturation magnetization in two opposite directions.As can be seen in figure 2, SPIONs exhibit magnetization values much higher than paramagnetic nanoparticles under a magnetic field of equal strength.However, without an external magnetic force, their innate magnetization is 0 (table 1) [15].Those properties are extremely important in the case of their medical applications-their magnetism can be 'turned on' only for a specific spatio-temporal window during imaging or therapy [13,16].The magneticity of SPIONs depends not only on their size but can also be controlled using appropriate additives or coatings [17][18][19].These properties make SPIONs highly promising for use in theranostics, both for diagnostic purposes and in the therapy of neoplastic diseases [20].They are able to form stable colloidal systems in biological and physiological matrices and, thus, may be multifunctional tools in cancer theranostics [21].As SPIONs based on the Fe 3 O 4 structure are the most commonly used for this purpose, the rest of this review focuses on them.

Fe 3 O 4 SPIONs theranostic applications
Certain Fe 3 O 4 SPIONs have already found application in medicine, and some of them have been approved for clinical use by the Food and Drug Administration (FDA) as contrasting agents in magnetic resonance imaging (MRI) [22,23].It is one of the most essential applications of SPIONs and is crucial to their use in theranostics.MRI is used to obtain images of the soft tissues within the human body containing water using an external magnetic field, and the undisputed advantage of SPIONs over previously used contrast modifiers (e.g. based on gadolinium) is non-toxicity [15].Application of the SPIONs modifies the tissues' relaxation times (T1 and T2), leading to better-quality images, and recent studies indicate the effect of the size and coating of synthesized SPIONs on obtained relaxation times [24,25].Those nanoparticles may be classified as the first generation of SPIONs, dedicated to diagnostics only.The currently performed research in the area is aimed primarily at designing a subsequent generation of SPIONs that would combine diagnostic functions with therapeutic ones and permit precise therapy adjustment for individual patients [26].
Fe 3 O 4 SPIONs have a wide range of potential applications following their earlier described properties [26][27][28].For instance, in photothermal therapy (PTT), they are used as nanocarriers of agents absorbing near-infrared radiation (NIR).These agents, irradiated by laser light, emit heat that kills cancer cells in a given area (tumour ablation caused by high temperature) [29,30].Moreover, when using an external variable magnetic field, the SPIONs may become a source of point hyperthermia [31,32].In these two applications, the generated heat leads to structural and functional damage of pathological cells that are less resistant to high temperatures than healthy cells.Even if temperatures produced are of an order of 42 °C, the neoplastic cells may be sensitized to radiotherapy or chemotherapy, permitting a reduction in the used dose of irradiation or drug and leading to alleviation of the side effects of the therapy [33,34].
Fe 3 O 4 SPIONs may also be successfully applied in chemotherapy for drug delivery as they are easily subjected to functionalization and, thus, can support different therapeutic substances bonded to the nanoparticles' surface [35].The use of SPIONs as nanocarriers permits the extension of time of the drug circulation in the blood, increasing the drug effectiveness and control of the drug release by different mechanisms, e.g.temperature, pH, or specific enzymes, which curbs their negative impact on healthy cells [26,36].The application of specific coatings can improve the adsorption of certain drugs (e.g.hydrophobic paclitaxel) and then target those nanoplatforms to particular cells and tissues within the human body [37].On the other hand, the combination of SPIONs with liposomes (magnetoliposomes) can be used for the controlled delivery and release of drugs, e.g.doxorubicin, a cytostatic drug commonly used to treat cancer, has been encapsulated within bilayer lipid membranes with the hydrophobic superparamagnetic nanoparticle.The SPIONs not only allowed for magnetically assisted drug delivery but were also used for controlled doxorubicin release based on the application of an alternating magnetic field, which could destroy liposomes due to local heating or mechanical disruption [38].
In photodynamic therapy, SPIONs may deliver photosensitizing agents that, when activated by irradiation of a specific wavelength, produce toxic reactive oxygen species (ROS), killing the cancer cells in the irradiated site [39,40].Another example is gene therapy in which SPIONs may act as carriers of nucleic acids that, through changing the genetic information of cancer cells (leading to their apoptosis), produce a desired therapeutic effect [41].Because of the low stability of nucleic acids, SPIONs, besides acting as carriers, also protect the transported nucleic acids against decomposition [27].Moreover, the use of an external magnetic field for the concentration of the nanoparticles at a certain site increases the effectiveness of this therapy [42].
It should be emphasized that a great advantage of SPIONs is the ability to combine different therapies on a single nanoplatform, which may enhance their effectiveness (figure 3) [31,36].Moreover, the use of MRI support permits localization and determination of the size and shape of the neoplastic change as well as a choice of the moment when the concentration of SPIONs in the tumour is the highest.It enables monitoring of the tumour and organism responses to a given therapy [29,30,39,40,43].This approach is assumed as the basis of theranostics and may be the future standard of anticancer therapies.

Synthesis of Fe 3 O 4 SPIONs
Potential biomedical applications of nanomaterials are closely related to their structure and composition.Synthesis of SPIONs capable of application in theranostics comprises three stages: synthesis of the nanoparticle core, core coating with a specific modifying agent protecting the core from the external environment, and functionalization of the nanoparticle surface to be able to attach specific ligands ensuring special therapeutic functions (figure 4) [27].

Fe 3 O 4 SPIONs core synthesis
Magnetic properties of SPION are determined by its core's composition, size and morphology.Synthesis of a nanoparticle of parameters endowing it with superparamagnetic properties is of key importance for the application in diagnostics and anticancer therapies.There are three types of SPION core synthesis: physical, chemical and biological (figure 5) [23].
Unfortunately, physical methods, including e.g.gas phase deposition, electron beam lithography, or laser-induced pyrolysis, do not permit precise control of the size of the Table 1.A comparison of properties between iron oxide and superparamagnetic iron oxide nanoparticles.

Iron oxide nanoparticles Superparamagnetic iron oxide nanoparticles
Size of the core <30 nm >30 nm Structure Multiple magnetic domains within the particle Single-domain particle Magnetic susceptibility Comparatively smaller Comparatively higher with rapid magnetic response Remanence and coercivity Negligible residual magnetization and coercivity Zero remanence and coercivity nanoparticles [44].Biological methods, based on the use of microorganisms or plant extracts, provide SPION cores of high biocompatibility.However, the yield of the processes is low, and the size distribution of the obtained cores is large [23,45].In view of the above drawbacks of physical and biological methods, the chemical ones are most often applied [46].Among them, for the synthesis of nanomaterials of theranostic applications, the most attractive are the methods of coprecipitation, sol-gel, microemulsion and thermal decomposition.The simplest and the most efficient among the chemical methods is that of coprecipitation of iron(II) and iron(III) ions in the presence of a base.It is one of the most economical ways to synthesize highly polydisperse SPIONs with satisfactory magnetic properties and low crystallinity due to its ease of implementation and large volume capacity [47][48][49].By adjusting experimental conditions (e.g.temperature, pH, stoichiometry) and adding various capping agents, SPIONs of various sizes and magnetic properties can be obtained [50].On the other hand, in microemulsion synthesis, where micellar microemulsion systems are created using a variety of amphiphilic surfactants, the size of SPIONs can be controlled using micelles of different sizes.Because of this, obtained nanoparticles are highly polydispersible [51].However, due to the low temperature of the reaction, relatively low crystallinity SPIONs are synthesized with worse efficiency.Another highly popular method, thermal decomposition, is characterized by SPIONs with great size and shape control, good crystallinity and narrow size distribution [52].However, the method is environmentally harmful due to the production of toxic substances (e.g.chloroform, hexane).Moreover, due to the SPIONs obtained with hydrophobic coatings, other steps and proper surface modifications are required in case of potential biomedical applications.In the case of the sol-gel method, which allows obtaining high yields of relatively large and monodisperse silica-coated SPIONs, additional purification is needed after synthesis due to contaminated by-products [53].Microemulsion and thermal decomposition methods permit better control of the size and morphology of SPIONs of core diameters below 20 nm [51,54].On the other side, the sol-gel method is preferred for obtaining nanoparticles of a size greater than 20 nm, which are to be coated with silicon coatings [55,56].

Fe 3 O 4 SPIONs surface modification
SPIONs stability in suspensions is determined by hydrophobic-hydrophilic and van der Waals interactions.The unmodified nanoparticles form agglomerates in order to reduce the high surface energy following from a high surface area to volume ratio, which at the same time restricts their superparamagnetic properties [57].While the SPIONs in low concentrations show very little to no toxicity, their aggregation or precipitation in saline can change it due to the increase in size [15].Modification of the SPIONs surface permits obtaining nanostructures stable in complex biological matrices and, thus, curbs the undesirable agglomeration processes, which can further lead to inflammation and other cytotoxic responses.Moreover, the modifying coatings may protect nanoparticles against oxidation, enhance their biocompatibility with human organisms and facilitate the attachment of functional ligands or drug molecules in the process of functionalization [23,57,58].It should be pointed out that when designing nanoparticles of potential biomedical applications, the modifier to be used should be non-toxic, biocompatible and biodegradable.A variety of coatings can be used for this purpose.Poly(ethylene)glycol (PEG) is one of the most common biocompatible polymers used to reduce SPIONs interactions with cells and proteins and enhance nanoparticles' blood half-time [15].Dextran, already used in Food and Drug Administration (FDA) approved Sinorem ® and Andorem ® , is used as the coating due to its amazing immunocompatibility with the innate immune system cells.As a consequence, dextran-coated SPIONs do not induce inflammatory monocyte reaction unless a high concentration is used [59].Chitosan, being another polysaccharide-based, highly biodegradable and biocompatible coating, has been shown to exhibit anticoagulant properties [60].Silica coatings not only increase the biocompatibility of SPIONS but also, due to their mesoporous structure, can be effectively used for controlled drug release, e.g.doxorubicin or poorly soluble exemestane [61].PVP (polyvinylpyrrolidone), an amphiphilic, non-toxic, and biocompatible polymer, stabilizes the SPIONs, reducing their aggregation and increasing their dispersibility in biological fluids, allowing for their use in enhanced T2-weighted MRI [62].LCA (conjugated linoleic acids, natural fatty acids) not only limit the agglomeration of SPIONs due to the presence of carboxylic groups but also exhibit anticancer activity by affecting the lipid metabolism pathways of tumour cells [37].

Functionalization of Fe 3 O 4 SPIONs surface
As mentioned above, the aim of SPIONs surface functionalization is to endow them with specific chemical properties and functions as a result of the attachment of selected ligands and biomolecules [63].The attachment of these compounds is realized by one of the following four mechanisms: (I) through chemisorption, e.g. using thiol groups; (II) through electrostatic adsorption of molecules of the opposite charge; (III) through covalent bond formation between certain functional groups and (IV) through non-covalent bond based on receptor-ligand affinity [64].According to the type of their activity, the functionalizing substances can be divided into two groups.The first one comprises compounds of therapeutical activity that are to be delivered by SPIONs to cancer cells in order to evoke a certain response leading to cancer cells' apoptosis or damage.This group includes drugs, nucleic acids and photosensitizers [35,36,[39][40][41].The second group are the compounds responsible for the targeted transport of nanoparticles to tumours.
Biodistribution of SPIONs in human organisms may proceed in two ways.The first is the passive transportation of nanoparticles to tumours using enhanced permeability and retention of nanoparticles in the tumour blood vessels relative to those in the healthy tissues (named as the EPR effect) [65].Similar phenomena occur in human organisms in the case of inflammations, which can reduce the effectiveness of the passive transportation of drugs to tumours [66].Moreover, of crucial importance in passive transportation is the choice of the modifying agent that would ensure an extended time of SPIONs circulation in the blood.The right choice of a modifier facilitates the nanoparticles' transportation to cancer cells before they are eliminated by the phagocytic system [65].The active transportation is determined by the right functionalization of nanoparticles surface with specific directing ligands that are able to recognize and then attach to target receptors on the surface of cancer cells [67].This process leads to enhanced accumulation of nanoparticles in the tumour and restricts the negative impact of particles transported by the nanoparticles on healthy tissues.A very interesting feature, unique for SPIONs, is the possibility of supporting the transportation process with an external magnetic field, which may also be an alternative to active transportation [68].In the case of potential cancer theranostic application, bio-inspired coatings deserve special attention, as they can further improve the stability of nanoparticles under physiological conditions, target SPIONs to specific sites in  the body, and promote the penetration of cancer cells and tissues [69].There are numerous examples of targeting moieties that can be used for this purpose: antibodies [70], aptamers [71], hyaluronic acid [72], folate [73], human serum proteins (albumin [74] and transferrin [75]) or various targeting peptides (e.g.H 7 K(R 2 ) 2 [76], ferritin [77], GE11 [78], RGD peptides [79]).

Characterization of physiochemical parameters of Fe 3 O 4 SPIONs
An increasing number of nanomaterials obtained by a wide range of modifications and surface functionalization is accompanied by a growing demand for analytical methods allowing precise characterization of their parameters.Such a characterization is of great importance in the synthesis of nanoparticles designed for specific biomedical applications.The nanomaterials should meet certain requirements depending on their future use.So far, several techniques mentioned below have been used to characterize the physicochemical properties of Fe 3 O 4 SPIONs [80][81][82].
X-ray diffraction (XRD) is based on the elastic scattering of x-rays on the nanomaterial studied to obtain a diffraction pattern that permits the determination of its crystalline structure (including the type of crystal lattice) and size of nanoparticles [84][85][86].Dynamic light scattering (DLS) enables evaluation of the size of SPIONs in suspensions and emulsions (determination of the hydrodynamic diameter of nanoparticles) based on the speed of nanoparticles performing Brownian motion [43,85].The information on ζ-potential (zeta potential), which describes the surface charge on SPIONs (strictly related to the nanomaterial stability), is obtained from measurements of electrophoretic mobility in an electric field [84].Microscopic techniques like scanning electron microscopy (SEM) and transmission electron microscopy (TEM) provide information on the morphology, core size and monodispersity of nanoparticles using a beam of electrons falling on the nanomaterial surface (SEM) or passing through the nanomaterial (TEM) [83,85,87] (figure 6).Fourier transform infrared spectroscopy (FTIR) permits the determination of the composition of the nanomaterial surface and its functionalization by identification of specific bonds between atoms (on the basis of absorption of infrared radiation of certain frequencies related to the energies of the bonds vibrations), while vibrating sample magnetometry (VSM) permits investigation of magnetic properties of SPIONs using the phenomenon of magnetic induction [84,89].

Protein corona
The successful design of nanomaterials of potential theranostic applications requires considering the processes they will be involved in and the transformations they will undergo after introducing them to the physiological environment.After exposition to the active components of human blood, SPIONs will be surrounded by the proteins from the blood serum, making the so-called protein corona [90].This process is initiated by a high surface energy of nanoparticles, leading to the adsorption of protein components on their surface.According to the currently assumed hypothesis, the protein corona is composed of two layers, a soft one and a hard one, differing in the strength of the proteins binding to the nanoparticle (figure 7) [88].
The corona formed in this way may stabilize SPIONs, limiting their possible agglomeration (figure 8) and is a crucial factor in phagocytosis processes [91,92].Immunoglobulins, which are one of the most numerous proteins forming the corona, are responsible for the stimulation of the immunological system to remove the nanoparticles [93].Modifying SPIONs surface with, e.g.PEG, restricts the adsorption of proteins through forming a steric barrier and, thus, protects the nanoparticles from opsonization.Unfortunately, the modifications applied are insufficient for the total elimination of protein corona formation [94].
The corona significantly affects the nanoparticles' interactions with their chemical environment [88].It is one of the reasons why the development of active transportation using SPIONs functionalized with directing ligands has stopped at the phase of pre-clinical tests [9].The protein corona may mask the molecules of the substance used for functionalization and thus block the interactions between the directing ligands and the surface of tumour cells.The formation of protein corona implies the need to consider the processes of protein adsorption on the nanoparticles' surface in designing SPIONs of specific theranostic applications.
According to the literature, there is a possibility of controlling the composition of the forming protein corona as it has been proved that there is a correlation between the type of Fe 3 O 4 SPIONs modifiers and this composition.Between bare, citrate-coated and riboflavin-coated SPIONs, the latter absorbed more serum proteins.The reason for this was most likely the higher hydrophobicity of riboflavin ligand, which promoted interactions of SPIONs with human serum components-both the number of distinct proteins and the overall protein amount were higher [87].Differences in corona composition were also observed between silica-coated and dextran-coated SPIONs.While for silica-modified SPIONs, fibrinogen alpha, beta, and gamma were the most abundant corona components, in case dextran-modified nanoparticles blood coagulation associated kininogen-1, microtubule-associated serine/threonine kinase-like and platelet factor 4 [95].Consequently, through the use of specific modifiers and modification of the nanoparticles' size and composition, it is possible to control the protein corona composition, as well as the SPIONs biodistribution in the organism [96].Moreover, it is worth noting that while the size of nanoparticles does not impact corona composition quantitatively, it affects the surface energy and, thus, the protein loading per particle-smaller nanoparticles adsorb more proteins on an area-normalized basis [97,98] Regarding surface chemistry behind the protein corona formation, the interactions between SPIONs and proteins are a broad subject due to the possibility of modifying the surface of nanoparticles, thus affecting the primary force behind the adsorption.However, the literature emphasizes the importance of physical adsorption processes-the interactions between nanoparticles and proteins can be considered a blend of hydrophobic effects, hydrogen bonding, electrostatic forces and van der Waals attraction [97,[99][100][101].On the other hand, the utilization of specific modifications can lead to different, more robust types of interactions, e.g.binding antibodies on the surface allows for selective antibody-antigen bonds or using covalent bonding between amine groups and specific reactive groups, such as aldehydes or epoxides, conjugated to SPIONs [102,103].
Also, worth emphasizing are attempts to use human blood serum proteins to coat the magnetic nanoparticles.This can result in changes in their stability, pharmacokinetics, and biodistribution within the human body, leading to more efficacious targeted delivery of anticancer drugs.Numerous studies are being conducted on the use of, e.g.albumin for this purpose, as it is the most common protein in human blood serum, and its use may lead to improved biocompatibility of the coated nanoparticles and extend their circulation time in the blood.Moreover, the functionality of those albumincoated nanoparticles can be enhanced by integrating different therapeutics in their structure using a covalent approach or albumin binding [74].On the other hand, the use of transferrin may be a potential solution to the problem of directional ligand masking by the corona.Transferrin is a blood serum protein responsible for targeted transportation of iron ions to cancer cells [75], which show overexpression of transferrin receptors because of increased demand for iron in carcinogenesis [104].Attempts at the application of transferrin as a directional ligand have already been made, although they did not bring expected results.The 'in vitro' studies have not shown significant differences in the accumulation of Fe 3 O 4 SPIONs functionalized with transferrin and those without this compound [105].A possible reason may be the functionalization method, which was made in laboratory conditions, and then the obtained functionalized nanoparticles were introduced to the physiological environment.It has been reported that the protein corona has a structure with a dynamically changing composition [106].The proteins accumulated on the SPIONs surface, including the initially attached transferrin, are replaced by other blood serum proteins that are either more numerous or show greater affinity to the nanoparticles.An interesting approach to solve this problem may be 'in situ' functionalization after SPIONs introduction to the human circulatory system.As mentioned above, it is possible to control the composition of a forming protein corona, which implies a hypothesis that a proper modification and functionalization of nanoparticles surface could lead to nanoparticles that, on contact with blood serum proteins, would selectively bind transferrin.However, to make attempts at obtaining such nanoparticles, it is necessary to have tools that would permit a detailed examination of the interactions between SPIONs and human serum proteins as well as the determination of mechanisms responsible for the formation of protein corona and its composition.Only this knowledge will allow making attempts at selective functionalization of Fe 3 O 4 SPIONs surface with transferrin in human organisms.

Characterization of Fe 3 O 4 SPIONs interactions with proteins
Preliminary evaluation of SPIONs properties and pre-clinical observation of changes in chemical species in biological matrices are necessary before their approval for medical use.However, the previous studies have been mainly focused on post-synthetic characterization of basic parameters of the obtained nanomaterials.Even if the problems of interactions with biological compounds have been considered, the studies have been limited to the simulation of interactions with one selected compound, disregarding the effects of the other components of the matrix (e.g.human serum proteins) on the changes taking place in the studied suspensions.Consequently, although a large variety of nanomaterials have been designed in the last decade, only a few have been approved for commercial use [107].For this reason, developing the right tools and methodologies that permit the investigation of SPIONs interactions with human serum components and characterization of nanomaterial changes on contact with biological matrices seems pivotal.The above-outlined area of research should include the following three thematical problems that should be addressed before the proposed nanomaterials can be admitted for clinical use [108].
The first range of problems concerns the composition of the forming protein corona.The primary method for determination of the protein corona composition is the 'bottom-up' proteomic analysis based on the determination of masses of the peptides obtained as a result of enzymatic digestion of the corona [109,110].In the 'shotgun' type proteomics, a composition of proteins is subjected to proteolytic digestion, and then the obtained peptides are separated using high-performance liquid chromatography (HPLC).It is also possible to use one-or two-dimensional polyacrylamide gel electrophoresis (PAGE) before HPLC [87].The obtained mixture of peptides is then analyzed by tandem mass spectrometry (MS/ MS) with electrospray ionization (ESI) or matrix-assisted laser desorption/ ionization (MALDI).The obtained data are compared with the information collected in databases to identify individual peptides and their sequences, as well as the proteins of the corona [96].An alternative, less complex, analytical technique providing preliminary information on the composition of the protein corona is sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).A sample of SPIONs with proteins is subjected to thermal denaturation, then to electrophoretic separation in the presence of a reducing compound, and based on the positions of bands in the electrophoretic pattern and their comparison with the database, the proteins present in the corona are identified [87,95,111].The magnetic properties of Fe 3 O 4 SPIONs are an undeniable advantage in the preparation of samples of the above type and separation of SPIONs connectivities with proteins from the unreacted proteins in both described above techniques [112].However, it should be emphasized that because of the dynamic character of the corona structure, it does not reach a state of equilibrium and analysis of its composition may bring different results even for the samples prepared in parallel [113].
The second range of problems that need to be addressed in the investigation of SPIONs with protein components concerns their stability in biological matrices.The abovedescribed techniques for characterization of the nanoparticles' surface can obtain information on this subject.Dynamic light scattering (DLS) and ζ-potential measurements permit the determination of changes in the nanoparticle surface caused by the formation of protein corona, determination of the hydrodynamic diameter of the corona and its surface charge, which brings information on the stability of the nanoparticles and their tendency to agglomerate [114,115].Changes in the Fe 3 O 4 SPIONs stability in protein presence may also be monitored by single particle inductively coupled plasmatandem mass spectrometry (spICP-MS/MS).This technique is not suitable for the determination of the size of nanoparticle cores because they are too small.However, it provides information on the effect of the protein corona formation on the limitation of nanoparticles agglomeration [91].An important question in this range of problems is the stability of the nanoparticles in matrices containing biological components.The compounds contained in blood serum may initiate the gradual release of iron ions, leading to a slow decomposition of nanoparticles.For this reason, it is important to check the stability of SPIONs in physiological salt buffered with phosphates and, if possible, also in the environment of undiluted human blood serum [116].For this purpose, literature has proposed the use of inductively coupled plasma mass spectroscopy with double focusing that permits monitoring of Fe 3 O 4 nanoparticles degradation on the basis of the released iron ions [117].
Another range of problems of key importance for designing nanoparticles of potential medical applications is the determination of the kinetics of processes taking place on the nanoparticles' surface upon their introduction to a biological matrix.Different proteins are characterized by different affinity towards the modified and functionalized SPIONs.It is assumed that the proteins with higher binding affinity to the nanoparticles will form a hard corona, while those with lower binding affinity will form a soft one [118].However, the processes and mechanisms involved in the formation of protein corona are not known as yet.According to one theory, directly after SPIONs' introduction to the physiological matrix, the dominant matrix components will adsorb on their surface at first, and then, with time, they will be replaced by the components showing higher binding affinity to SPIONs.According to another scenario, the kinetically preferred proteins will be adsorbed at first and with time, they will be replaced by those present in dominant abundance (e.g.albumins) [108].
Determination of the kinetics of processes that SPIONs are involved in after introduction to the human circulatory system is vital because of the significance of time in the diagnostic and therapeutic processes with the use of nanoparticles.Investigation of SPIONs should consider the time of their circulation in human organisms and changes in the protein corona at that time.
The previously mentioned techniques may be used to study the kinetics of processes on the SPIONs surface upon their introduction to the physiological matrix.In literature reports, the use of SDS-PAGE and FTIR for monitoring the effect of the incubation time on the composition of the formed protein corona has been described [119].A significant effect of incubation time on the hydrodynamic diameter and ζpotential, whose values changed with gradual modification of the protein corona composition, has also been implicated [111].Monitoring changes in the content of sulfur and certain metals contained in the metalloproteins incorporated in the corona structure has been realized with inductively coupled plasma mass spectroscopy (ICP-MS) [120].

Hyphenated techniques in investigation of Fe 3 O 4 SPIONs interactions with proteins
The above-described techniques are insufficient to provide data allowing the elucidation of protein adsorption on the nanoparticles' surface and the mechanisms of the processes behind this phenomenon.In order to disclose the principles governing the binding affinity of individual proteins to proposed Fe 3 O 4 SPIONs, it is necessary to have tools that would permit simultaneous separation and detection of different SPIONs species forming in samples containing components of human serum proteins.A potential response to the above demand may be hyphenated techniques.The binding between SPOINs and human serum proteins may be characterized by the application of high-performance techniques for species separation, such as capillary electrophoresis (CE) or HPLC coupled with sensitive modules for their identification and determination [121,122].

Capillary electrophoresis and high-performance liquid chromatography
CE and HPLC are commonly used to separate proteins for their subsequent identification by appropriate detectors [123][124][125].However, the literature provides only a few examples of separation techniques for investigating Fe 3 O 4 SPIONs.So far, only the attempts at separation of SPIONs of different surface modifications by capillary electrophoresis have been successful [126].There are also reports describing the use of capillary zone electrophoresis to study nanometric structures of iron oxide.A significant impact of the nanomaterial surface charge on the nanoparticle's adsorption to the capillary internal surface has been reported.Thus, it has been recommended to modify the nanoparticles' surface with certain modifiers like tetramethyl citrate of hydroxide (TMAOH) in order to change their surface charge into negative or to modify the internal surface of the capillary with a cationic surfactant [127,128].
Moreover, it has been emphasized many times that when using capillary electrophoresis, the impact of the buffer composition on the quality of signals coming from SPIONs should be considered [129,130].In literature reports, capillary electrophoresis has been combined with UV/Vis detectors of diode array detector (DAD).Much fewer examples of the use of HPLC for the separation of magnetic nanoparticles have been given.This technique has been applied in investigating the metabolites of iron oxide nanoparticles forming after incubating the nanoparticles in simulated gastric acid conditions and in cell cultures (Caco-2 and HT-29).In the latter experiment, the use of HPLC permitted the separation of different iron species (monodisperse nanoparticles, their aggregations and ionic species of iron) [131].If the study aims to simulate physiological conditions and a possible 'in situ' functionalization, capillary electrophoresis seems to be a better choice.In this process, separation takes place in mild conditions of a physiological buffer, in contrast to HPLC, which often requires the use of organic solvents as a mobile phase.
Moreover, HPLC needs surfactants to elute the nanomaterials from the column, and these compounds may disturb the formation of protein corona around the nanoparticles [132].Other advantages of capillary electrophoresis are related to smaller sample volume and higher resolution compared to HPLC application.This is important for separating different SPION species forming in human blood serum.Another important feature of CE is that in its mechanism of separation, the analytes do not interact with elements of the system, e.g. the column packing in HPLC, so the impact of the system on the forming protein corona should be limited.

Inductively coupled plasma tandem mass spectrometry (ICP-MS/MS)
Inductively coupled plasma mass spectrometry is one of the leading techniques in elemental analysis.It permits the determination and identification of metals and metalloids in ultra-trace amounts in diverse matrices with high sensitivity and low detection limits [133].ICP-MS has been applied in the investigation of SPIONs, e.g. for the determination of the content of iron in nanoparticles accumulated in mouse or rat organs or in selected strains of cancer cells [63,134,135].It should be mentioned that analysis of 56 Fe (the most abundant iron isotope) by ICP-MS is charged with the presence of polyatomic spectral interferences, e.g. from 40 Ar 16 O originating from the gas used for maintaining the plasma flame and 40 Ca 16 O coming from the matrix components [136].The use of tandem mass spectrometry (MS/MS) is one of the strategies aimed at limiting the above effects through the employment of a collision/reaction cell that minimizes the impact of the spectral interferences on the result of measurement [137].An important point that must be considered when applying the above-described technique for the investigation of SPIONs interaction with proteins, is the correct choice of the collision/reaction gas, which should be made not only to ensure minimization of interferences to the iron signal but also to generate the signal of sulfur being a component of cysteine and methionine (the amino acids occurring in proteins) [138].Determination of low concentrations of sulfur (as the most abundant isotope of 32 S) has been impossible for a long time because of polyatomic interferences (including those from 32 O 2 ).Only the use of a collision/reaction cell with oxygen as a reaction gas permitted monitoring of sulfur signal thanks to the change in the mass-to-charge ratio of the analyte ( 32 S 16 O) [139].

Capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS)
Coupling the modules for the hyphenated technique CE-ICP-MS/MS requires using a unique interface based on cross-connection.The interface permits sample supply with the so-called sheath liquid, i.e. a diluted buffer solution with an internal standard addition.This solution, on the one hand, allows the closing of the electric circuit in the capillary electrophoresis process and, on the other hand, enables effective transportation of small volumes of the sample to the source of ions after the preliminary separation of the sample in the capillary tube.At first, the hyphenated technique of CE-ICP-MS/MS was used for the investigation of such nanomaterials as gold nanoparticles and quantum dots, permitting monitoring of their interactions with human serum proteins [140,141].In order to be applied for the investigation of Fe 3 O 4 SPIONs, the CE-ICP-MS/MS method has been optimized so that it would permit a simultaneous determination of sulfur and iron (protein and nanoparticle markers, figure 9) [142].Later research works have shown a significant effect of the magnetic properties of the nanoparticles on the stability of protein corona formed on the SPIONs surface in the conditions of electrophoretic separation in the case of synthesized SPIONs, which exhibited greater magneticity than commercially available ones (figure 10) [143].

Conclusions
In this review, the authors introduced the SPION-scientific beginners with the arcane of their properties versus analytical methodology for investigating their behaviour in the presence of proteins.The pros and cons of Fe 3 O 4 SPIONs application in theranostics were pointed out.On one side, it must be stressed that the synthesized growing number of novel Fe 3 O 4 SPIONs has not yet been translated into the number of solutions approved for clinical use.On the other side, it can be related to the lack of proper tools that could permit the examination of these nanomaterials with human blood serum proteins.It needs to be emphasized that such investigations are vital for elucidation of the changes in the structure and properties of the designed SPIONs in the physiological environment to be able to have significant control on the effectiveness of clinical tests through the choice of specific parameters of the nanoparticles.After the lecture, it becomes evident that SPIONs themselves can be treated as analytical challenge.For instance, the magnetic properties of SPIONs are attractive given their potential applications but may considerably hinder analytical studies.The presented review highlighted the need to design and develop new methodologies for investigating SPIONs interaction with human blood serum proteins that would consider the specific features of nanoparticles of potential applications in medicine.

Figure 1 .
Figure 1.Types of different nanomaterials structures commonly used in anticancer therapies.

Figure 2 .
Figure 2. Hysteresis curve representing the relationship between magnetization value and applied magnetic field for the diamagnetic, ferromagnetic, paramagnetic, and superparamagnetic particles.Reproduced from [15].CC BY 4.0.

Figure 4 .
Figure 4. Schematic of structure of an exemplary multilayered SPION with biocompatible and therapeutic coatings designed for theranostic applications [27].

Figure 7 .
Figure 7. Schematic representation of the protein corona divided into hard and soft corona according to the currently assumed hypothesis.Reproduced from [88].CC BY 4.0.