Nonlinear Magnetochemical Effects in Nanotherapy of Walker-256

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NONLINEAR MAGNETOCHEMICAL EFFECTS in NANOTHERAPY of WALKER-256 CARCINOSARCOMA Valerii Orel, Marina Tselepi, Thanos Mitrelias, Mykhailo Zabolotny, Mykhailo Krotevich, Anatoliy Shevchenko, Alexander Rykhalskyi, Andriy Romanov, Valerii B. Orel, Anatoliy Burlaka, Sergey Lukin, Vladyslav Stegnii, and Crispin Barnes ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00526 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Applied Bio Materials

NONLINEAR MAGNETOCHEMICAL EFFECTS in NANOTHERAPY of WALKER-256 CARCINOSARCOMA Valerii E. Orel1 ,2, *, Marina Tselepi3 ,4, Thanos Mitrelias3, Mykhailo Zabolotny5, Mykhailo Krotevich1, Anatoliy Shevchenko6, Alexander Rykhalskyi1, Andriy Romanov1, Valerii В. Orel7, Anatoliy Burlaka8, Sergey Lukin8, Vladyslav Stegnii2 and Crispin H.W. Barnes3

1

National Cancer Institute, Kyiv, Ukraine

2

Biomedical Engineering Department, NTUU “Igor Sikorsky KPI”, Kyiv, Ukraine

3

Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom

4

Department of Physics, University of Ioannina, Ioannina, Greece

5

Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

6

G.V. Kurdyumov Institute for Metal Physics, Kyiv, Ukraine

ACS Paragon Plus Environment

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ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Bogomolets National Medical University, Kyiv, Ukraine

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R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, Kyiv,

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Ukraine

*Corresponding author: Prof. V.E. Orel National Cancer Institute, 33/43 Lomonosov St., 03022, Kyiv, Ukraine. E-mail: [email protected]

KEYWORDS: magnetochemistry, oxidative stress, inhomogeneous magnetic field, tumor heterogeneity, cancer.

ABSTRACT. The biological and medical aspects of magnetochemical effects in nanotherapy of tumors remain poorly studied. The present paper investigates the influence of nonlinear magnetochemical effects of anisotropic magnetic nanodots on an animal tumor model. The magnetic properties and electron spin resonance spectra of magnetic nanodots and doxorubicin were investigated after mechano-magneto-chemical synthesis. The results obtained from the analysis of nonlinear kinetics and survival in Walker-256 carcinosarcoma bearing animals found a nonlinear dependence between the


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value of the growth factor, braking ratio, survival rate, tumor redox state and the treatment by the magnetic nanodot combined with a non-uniform constant magnetic field. To quantify the heterogeneity in microphotographs of Walker-256 carcinosarcoma sections, we applied the entropy parameter. The control (no treatment) group showed the greatest heterogeneity. The lowest value of tumor heterogeneity among animals given treatment was found in groups with the minimum growth factor. Similarly, the lowest entropy value was found in muscle tissue taken from inoculation areas of the tumor. The evidence from this study concluded that inhomogeneous constant magnetic fields with different strength applied to heterogeneous tumor tissues induced different magnetic anisotropy in the magnetic nanodot which had a significant influence on the nonlinear kinetics, redox state and histological pattern of the tumor.

Introduction. The most recent estimate suggests that 18 million new cancer cases were diagnosed in 2018. Cancer took the life of 9.6 million people and nearly one in six deaths were attributed to cancer worldwide. The disease has a dramatic impact on the quality of life and causes a substantial financial burden to patients, healthcare systems and society.


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This indicates a need to reduce the global cancer burden by improving the treatment outcome.1 Cancer starts with chaotic and poorly controlled changes in cell growth. In the human host, the stochastic nature of cell signaling has a major influence on cancer development, generally seen as a more important contributor than genetic or environmental factors. With the existing knowledge, cancer bears the hallmarks of a complex adaptive system. Changes in cell shape during carcinogenesis are an example of tumor adaptation. Cancer is observed to have a key property of a dynamical system which can be perturbated by host and treatment responses. The emerging role of the nonlinear dynamics in cancer has offered the concept of deterministic chaos that functions within the host hierarchy. A fundamental approach to tackle the issue of chaos relevance to cancer is to modulate the malignant process from hierarchical chaotic growth to regulated order.2,3 Over the past few decades, there has been an increasing interest in the use of nanotechnology in numerous biomedical applications such as magnetic drug delivery and targeting due to their ability to alter the pharmacokinetics of medication and affect nonlinear tumor growth kinetics. Particular emphasis was given to nanostructures used


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to initiate redox responses in a nonlinear manner.4 The application of a spatially inhomogeneous magnetic field with different gradients to biological systems can act on the cell function and even more the whole organism. Functional changes occur through several mechanisms, one of which involves a change in the probability of a voltage-gated ionic channel being open or closed under the influence of the magnetic field. At the nanoscale, the applied magnetic gradients can significantly vary depending on the distance between electrons in a free radical pair and thus regulate the cellular redox state.5 G. Lewis is probably best known for his magnetochemical theory presented in 1923. Later he investigated6 the magnetic properties of oxygen in solution in liquid nitrogen.7 The magnetochemical theory associated the valence theory and the formation of electrons between atoms representing a covalent bond. The oxygen and nitric oxide molecules have two 3-electron bonds and one 2-electron bond, whose ground-states are paramagnetic.8 Magnetochemical effects and redox reactions of metal-organic complexes are a crucial part in a series of vital processes.9


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Previous research carried out on Walker-256 carcinosarcoma and Lewis lung carcinoma with hematogenous metastases from a primary tumor has reported that a magnetic nanodot (MN) with the radio-frequency controlled magnetic memory effect can be exploited for nanomagnetic modulation of the tumor redox state in the course of anticancer therapy. Nanomagnetic modulation is based on a modiﬁed core-shell structure, in which a competition arises between the effects of spin canting and site exchange of free radicals located on the surface shell of a ferromagnetic nanodot.10 In addition, there is a notable difference between nanoscale and bulk ferromagnets in their behavior during nonlinear interaction and excitation. Therefore, nonlinear magnetization dynamics can characterize ferromagnetic nanodots.11 The above-mentioned nonlinear magnetic properties could play a decisive role in nonequilibrium biological phenomena12 for nonlinear redox reactions and tumor growth kinetics under the conditions of electromagnetic irradiation.13 Naturally, chaotic dynamics is induced by redox perturbations arising from oxidative stress and can lead to carcinogenesis. Disturbances in the balance between antioxidant defense and reactive oxygen species (ROS) and nitrogen species (RNS), collectively


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referred to as RONS, generated by the respiratory chain can trigger chaotic dynamics in mitochondrial function. A greater number of RONS are molecules containing an unpaired electron in their outer shell. RONS are formed endogenously as a by-product of cellular respiration in mitochondria or synthesized by enzymes involved in xenobiotic metabolism.14 Mitochondria undertake principal functions such as ATP production, maintaining redox balance, regulation of apoptosis and so these organelles are involved in the development of human diseases.15 On top of that, RONS are essential for modulation of cellular signaling in mammalian physiology. Traditionally, redox modulation of tumors has been thought to have significant therapeutic implications.16 Recent advances in nanomagnetic modulation of the cellular redox state are considered to be a promising therapeutic approach for cancer treatment. From this point, cancer cell death is induced by a synergistic combination of pro-oxidants (redox active compounds) and non-redox active compounds which modulate RONS formation, overcoming either chemotherapy resistance or toxicity associated with high doses of single drug use. Moreover, the dose of each compound is reduced in such a combination.17, 18


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As reviewed in the above-cited literature, RONS mediated cell sensing and metabolism are strictly regulated. Hence, it could conceivably be hypothesized that low (basal) levels of RONS promote cell growth and differentiation, whereas an increase in RONS is responsible for pathological phenotypes and genetic instability leading to cytotoxic effects and, furthermore, cell death. RONS are known to influence the nonlinear tumor growth kinetics.19 However, the experimental evidence for the relationship between the nonlinear magnetochemical effects of a redox modulating technology and nonlinear tumor growth kinetics is still insufficient to improve treatment outcomes for cancer patients. The current paper is a continuation of our previous work20 and will investigate the influence of a local inhomogeneous constant magnetic field (CMF) based on magnetochemical effects upon the nonlinear tumor growth kinetics. Results

Nonlinear kinetics and survival of Walker-256 carcinosarcoma

Previous studies have reported that the antitumor effect of MN on its own without external irradiation of Walker-256 carcinosarcoma is comparable with the action of


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conventional doxorubicin (DOXO). Therefore, we investigated the influence of MN exposed to different CMF on nonlinear kinetics of Walker-256 carcinosarcoma. Fig. 1 and Table 1 show the combined effect of MN and different CMF on the growth kinetics of Walker 256 carcinosarcoma from the 7th to the 21st day after tumor implantation. There was a significant difference in the tumor growth kinetics between the control group and treatment by conventional DOXO, MN + magnetic-dipole applicator (MDA) 1, MN + MDA 2 and MN+ MDA 3. Based on the braking ratio κ, the findings revealed that MN + MDA 2 had caused the maximum antitumor effect compared to either MN + MDA 1, MN + MDA 3 or conventional DOXO. MN + MDA 2 treatment demonstrated the highest tendency to survive (70%) among tumor-bearing animals on the 90th day after tumor implantation (Fig. 2). 30% of the tumors also became impalpable in the fourth group of animals. 90 days after tumor implantation muscle biopsies were taken from the inoculation areas for histopathological examination.


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Table 1. The growth kinetics on the 21st day after Walker-256 carcinosarcoma implantation

Group

Experiment

Parameters

of animals Growth factor

Braking ratio κ

φ, day–1

1

Control (no treatment)

0.68 ± 0.01

1.00

2

Conventional DOXO

0.52 ± 0.01*

1.32

3

MN + MDA 1

0.46 ± 0.01*+

1.48

4

MN + MDA 2

0.34 ± 0.01*+#

1.98

5

MN + MDA 3

0.49 ± 0.01*+&

1.38

*

Statistically significant difference from control group, p < 0.05;

+

Statistically significant difference from conventional DOXO, p < 0.05;

#

Statistically significant difference from MN + MDA 1, p < 0.05;

& Statistically

significant difference from MN + MDA 2, p < 0.05.


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Figure 1. The growth kinetics of Walker-256 carcinosarcoma (M ± m): 1– control (no treatment); 2 – conventional DOXO; 3 – MN + MDA 1; 4 – MN + MDA 2; 5– MN + MDA 3.

Figure 2. Kaplan-Meier curves displaying survival rates of animals on the 90th day after Walker-256 carcinosarcoma implantation: 1 – сontrol (no treatment); 2 – DOXO; 3 – MN


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+ MDA 1; 4 – MN + MDA 2; 5 - MN + MDA 3. All survived: *p < 0.01; versus сontrol (no treatment); using the log-rank test.

Histopathologic examination of the tumor

Figure 3. Histological sections of Walker-256 carcinosarcoma (H&E × 100) and the image entropy (E, arb. units): 1– control (no treatment) (E = 5.36); 2 – conventional DOXO (E = 5.30); 3 – MN + MDA 1 (E = 5.14); 4 – MN + MDA 2 (E = 5.14); 5 – MN + MDA 3 (E = 5.27); 6 – MN + MDA 2 (the muscle) (E = 4.85).


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Histopathological image analysis was required to illustrate the changes in tumor tissue. Fig. 3 provides hematoxylin and eosin (H&E) stained tumor sections after conventional DOXO treatment and MN in combination with different CMF. Microscopic analysis revealed the presence of extensive spontaneous tumor necrosis in the first (control) group which was not given any treatment. Tumors collected from the second group (conventional DOXO treatment) showed < 30% focal necrosis, whereas few remaining muscle fibers appeared different from the tumor tissue with a mild form of dystrophic changes and absence of necrosis in the third (MN + MDA 1) group. After MN + MDA 2 treatment, group 4 tumors displayed necrosis, edema with vascular thrombosis, tumor cells undergoing dystrophic changes and loose cell-cell junctions. The combined effect of MN + MDA 3 (group 5) led to prominent foci of necrosis in tumors. It is important to note that tumor cell debris with focal dystrophic changes in the immediate vicinity of muscle fibers undergoing focal sclerotic changes and interstitial edema within the surrounding intact fibers accompanied by some loss of nuclei were visible in the muscle sections of group 4 animals with no palpable tumors on the 90th day after implantation. Naturally, MN are not visualized at the current magnification.


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Heterogeneity observed in microphotographs of Walker-256 carcinosarcoma sections was calculated from the entropy. As can be seen from Fig. 3, the control (no treatment) group had the greatest heterogeneity. The lowest value of tumor heterogeneity among animals given treatment was found in the 3rd and 4th groups which was in agreement with the growth factor φ from Table.1. Likewise, the minimum value of entropy was found in images taken from the muscle. Electron spin resonance spectroscopy

Figure 4. Electron spin resonance spectra of Walker-256 carcinosarcoma on the 21st day after tumor implantation. 1 – control (no treatment); 2 – conventional DOXO; 3 – MN + MDA 1; 4 – MN + MDA 2; 5 – MN + MDA 3; 6 – MN + MDA 2 (the muscle). Т = 77 К.


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Figure 5. The redox state in Walker-256 carcinosarcoma on 19th day after tumor implantation.

*

Statistically significant difference from control group, p < 0.05;

+

Statistically significant difference from conventional DOXO, p < 0.05;

#


&


●


Fig. 4 and 5 present the summary statistics for the analysis of the electron spin resonance (ESR) spectra from tumor and muscle tissue. The level of free iron in tumors collected from the 2nd to 5th groups after magnetic nanotherapy was on average about


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12 times the level of the 1st group and 7 times the level of the muscle. Although, the lowest level of free iron among treatment groups was reported in the 4th group along with the muscle. Lactoferrin is an iron-binding protein of the transferrin family that facilitates iron transport into cells and regulates serum iron levels and external secretions.21 In this study, lactoferrin levels were 37% lower in DOXO therapy than in the control. As for the remaining groups, the protein was 1.25 to 4.6 times elevated compared to the control. Interestingly, lactoferrin levels in the 4th group and the muscle were almost equal. Ironsulfur clusters (NO-FeS) are proteins that contain iron atom liganded to inorganic sulfide with two, three or four iron centers in the protein. NO-FeS proteins play a central role in the electron transport chain and are only stable in a mixed valence state when at least one Fe2+ and one Fe3+ ions are both present.22 Tumor samples collected from MN + MDA groups had on average 1.6 and 3.4 times higher levels of NO-FeS-proteins than the control (1st group) and the muscle, respectively. Ubisemiquinone is a free radical produced by dehydrogenases in a single electron transfer of the mitochondrial respiratory chain. The unstable nature of ubisemiquinone allows it to be one of the major endogenous


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sources of ROS generation.23 Our study results indicated that the level of ubisemiquinone after MN + MDA 3 treatment was as four times high as in the muscle. Discussion

Unfortunately, the biological and medical aspects of magnetochemical effects in nanotherapy remain poorly discussed. Hence, the above-mentioned experimental studies provide theoretical bases for understanding the combined effect of MN and an inhomogeneous CMF on the nonlinear kinetics of Walker-256 carcinosarcoma, redox biochemical reactions and tumor histology. This section explains the findings which emerged from statistical analysis as well as develops a model to describe the mechanism of action of MN during magnetic nanotherapy in Fig 6.


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Figure 6. A suggested mechanism to explain magnetochemical effects of MN and a local nonlinear CMF on the inner mitochondrial membrane in malignant tumor cells: cellular redox potential (A), magnetic properties and conductivity (B), electrochemical gradient (C), electron and proton spin-dependent transport regulation leading to oxidative stress (D). First of all, MN comprised both Fe3O4 (magnetite) nanoparticles (NP) and DOXO. In the current investigation, MN had the properties of a soft ferromagnet, while DOXO as a component of MN acquired the characteristics of paramagnetic materials after magneto-


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mechano-chemical synthesis (MMCS). As is well-known, magnetite core-shell NP are semimetal magnetic semiconductors with a band gap at 0.14 eV. In other words, they exhibit both ferromagnetic and semiconductor properties at the surface (up to 3.4 % of atoms in a 50 nm NP are placed on the NP surface). Besides the total number of surface atoms responsible for the interaction, variations in the lattice structure of core-shell NP may be the reason why physico-chemical properties of surface atoms are different from those of the bulk. The latter favors the electron-hole recombination, i.e. when an electron in the conduction band recombines with a hole in the valence band, giving rise to free radicals in reactions between NP shell and the surrounding solvent. More importantly, the conduction band of Fe3O4 NP lies below the range of the cellular redox potentials -4.12 to -4.84 eV, which therefore enables electron transfer from biological redox couples to a range of energy levels in the conduction band (Fig. 6A). Redox couples observed in biological systems maintain the cellular redox state, however, the interaction with NP and subsequent electron transfer reactions lead to ROS generation, alteration in the prooxidant-antioxidant balance in favor of oxidative stress. Alternatively, electrons can be excited from the valence to the conduction band by applying the magnetic field in a


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semiconductor. The promotion of an electron leaves behind a hole in the valence band.24, 25

The holes on the metal oxide surface are strong oxidants and as a result of a hole

reaction with organic surface molecules the hydroxyl radical •OH can be generated, whereas the electrons mediate redox reactions by superoxide (•O2-) formation.26 The results obtained from the analysis of nonlinear kinetics and survival in Walker-256 carcinosarcoma bearing animals demonstrated that the lowest value of the growth factor φ and the highest braking ratio κ, as well as the maximum survival rate tendency, were found in the fourth group of animals given treatment by MN with the MDA 2 magnetic-dipole applicator. MDA 2 demonstrated the lowest average brightness level and the highest value of the entropy parameter after computer-aided analysis of particle alignment photographs. In our experiments, DOXO was used as an active compound of MN. DOXO is among the most commonly used anthracycline antibiotics to treat both hematogenous malignancies and solid tumors. One of the mechanisms by which DOXO acts involves ROS and RNS production resulting from redox cycling. DOXO redox cycling contributes to the DNA damaging in the cell nucleus and gives a more reasonable explanation for the antineoplastic activity of the drug than intercalation into DNA 27. Given a closer examination of magnetochemical processes induced by MN, it is now necessary to address magnetochemical effects of MN in combination with CMF on the tumor considering electron and proton spin-dependent transport regulation in mitochondria (Fig. 6 B). Previous research has reported that weak magnetic fields had a significant influence


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on redox reactions between biomolecules. The effect of CMF is explained by the conversion of unpaired electrons in radical pairs from the triplet to the singlet spin state. Similarly, this theory supports the redox mechanisms under investigation in experiments of ESR.28 A great deal of attention must be focused on the magnetic field gradient which creates an electrochemical gradient in the tumor cells. Once the cells have been exposed to a gradient magnetic field, molecules or atoms with an unpaired electron, such as free radicals and ions, are drawn towards regions of the higher field, assisting their movement across the cell membrane. According to the chemiosmotic hypothesis of P. Mitchell, oxidation of substrates in the respiratory chain causes protons (H+) to be transported from the inner mitochondrial matrix to the intermembrane space, generating an electrochemical proton gradient.29 The electrochemical gradient (H+-potential) across the inner membrane drives ATP synthesis during oxidative phosphorylation as protons move down the gradient through the ATPsynthases. Occasionally, electrons leak out of the respiratory chain giving rise to the production of •O2-, •OH. The electrochemical gradient is of the greatest importance in the nature of redox reactions in cancer cells and influence most cellular functions, such as differentiation,


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proliferation, apoptosis and necrosis.30, 31 Since the results of our study demonstrated a nonlinear antitumor response to inhomogeneous magnetic irradiation by CMF of different strength which directed spatial alignment of ferromagnetic NP, the observed nonlinear magnetochemical effects could be attributed to nonlinear modulation of a mitochondrial electrochemical gradient (Fig. 6 C). Cytochrome P450 hemoproteins are major enzymes involved in xenobiotic metabolism in the mitochondrial electron transport chain. Transitions between the low-spin and high-spin states in the heme group affect water dissociation, coordination and thus conformational dynamics at the active site of these oxidases. Disorder of the electron and proton spin-dependent transport can be adopted as a remote and local approach to regulate redox reactions in mitochondria. The presence of CMF enhances the antitumor activity of MN, for instance, it induces splitting of the electron energy levels in the course of a redox reaction producing superoxide or hydrogen peroxide by MN. For this reason, regulation of electron and proton spin-dependent transport can be employed to nonlinearly modulate tumor redox state initiating changes in tumor growth and tumor cell fate (Fig. 6 D). Apart from the chemiosmosis theory, several models attempted to shed some light on the mechanism of oxidative phosphorylation. P. Boyer has proposed that conformational changes are coupled with ATP synthesis, wherein electron transport at some protein structures could serve in the energy transmission from conformational states to oxidative phosphorylation.32 In addition, the mechano-chemiosmotic model has explained the impact of electromagnetic fields on electron and proton transport coupled with ATP


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production in energy transformation at the mitochondrial membrane.33, 34 Hence, the next part of the discussion moves on to describe in greater detail the proposed mechanism.

The magnetic moment of NP is given by p = MsV, (1) where Ms and V are the saturation magnetization and NP volume.

NP is often represented as a small, spherical magnet with the diameter equal to 2R, that is, a domain of magnetic NP acts as a dipole with the magnetic moment p. The magnetic induction B and the gradient located on the axis parallel to the magnetic moment direction are given by

𝐵// =

2𝜇0𝑀𝑠𝑅3 3𝑟3

2𝜇0𝑀𝑠𝑅3 and = , (2) 𝑑𝑟 𝑟4 𝑑𝐵//

Near the surface of the magnetic NP, when r = R, the modulus of the radial magnetic gradient is dB///dr = 2μ0Ms/R, as follows from (4). The perpendicular component (B⊥) is twice smaller than B//. Consequently, the magnetic gradient has the same order of


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magnitude due to the geometry of a magnet close to the surface: |B|  μ0Mr/r, where r is the characteristic length that defines the scale of a system. While the magnetic gradient force 𝐹 is applied to tumor cells, it can assist the transmembrane movement of free radicals and ions. 𝑑𝐵

𝐹 = 𝑝 𝑑𝑙 , (3) where p is the magnetic dipole moment of an ion, 𝐵 is the magnetic induction, and the derivative is taken with respect to direction l, which is parallel to the magnetic dipole moment of the ion, l//p.5 The spin-orbit interaction of electrons in NP generate magnetic anisotropy. Tumor tissue also exhibits the magnetic anisotropy energy and thus can influence MN clustering. In order to describe processes involving anisotropic structures, we used the same method as in

35, 36

assuming that the only region of a cluster that underwent chemical reaction

was the sphere with a volume of V. The simplest example of spherical symmetry in a cluster can be described:

V  4R 2  , (4) where R is the radius of a reaction surface, Δ is the width of the reaction sphere. If τ is the time taken for the surrounding structures to be present in the volume, then the velocity constant (k) of the surrounding structures attaching to a cluster:


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k

4R 2 



, (5)

where ϒ is the effective factor of a sphere that depends on the magnetic moment and the magnetic field strength. The final ratio relates R and the effective size of a cluster (Reff)

Reff 

3  R , (6) 16

A renormalized radius of the interaction may be interpreted as a result of a change in the effective interaction between the cluster and surrounding structures due to their anisotropy. The relationship (8) confirms that a reaction rate constant can be controlled by anisotropic magnetic clusters and codification of ϒ dependent on the magnetic field strength ( H ). Charge carrier motion in Fe3O4 magnetic semiconductors arises from diffusion driven by a concentration gradient of charged particles. In this instance, the diffusion current density is in proportion to the electrochemical potentials χn and χp expressed in terms of a one-dimensional model by equations 7 and 8: 𝑑𝜒𝑝

𝑑𝑝

𝑑𝜒𝑛

𝑑𝑛

(𝑗𝑝)𝑑𝑖𝑓 = ―𝑞𝑝𝜇𝑝 𝑑𝑥 = ―𝑞𝐷𝑝 𝑑𝑥 , (7) (𝑗𝑛)𝑑𝑖𝑓 = ―𝑞𝑛𝜇𝑛 𝑑𝑥 = ―𝑞𝐷𝑛𝑑𝑥 , (8)


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where q is the elementary charge; p, n – the hole and electron concentration; μ p, μn – the hole and electron mobility; Dp, Dn – diffusion coefficients for holes and electrons. As far as the mechanism of antitumor activity of MN is based on oxidative stress phenomena, the role of iron-dependent redox pathways is discussed below.13 Not only is the molecular oxygen used in signaling for normal cells, but it is also thought of as a potentially toxic agent for tumor cells contributing to many damaging intermediates and derivatives, commonly known as ROS (e.g., superoxide anion radicals •O2-, hydrogen peroxide H2O2, hydroxyl radicals •OH, peroxyl radicals, singlet oxygen). Elevated levels of toxic oxygen metabolites or ROS cause damage to lipids, proteins, DNA and carbohydrates leading to oxidative stress.37 The possible mechanism behind increased ROS production, namely hydroxyl radicals, in the presence of magnetite NP can be explained by Fenton and Haber–Weiss chemistry that drives hydroxyl radical generation from superoxide and hydrogen peroxide: Fe2+ complex + H2O2 → Fe3+ complex + •OH + HO¯ (9) Fe3+ complex + •O2- → Fe2+ complex+ O2 (10)


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The heterolytic cleavage of peroxide (O-O bond) in the presence of Fe3O4 NP forms superoxide anion radicals and thus is associated with oxidative stress (reactions 11, 12): (Fe3+) + ROOH → (FeO)3+ + ROH (11) (FeO)3+ + ROOH → (Fe3+) + ROH + 1O2 (12) High valent iron can also catalyze the heterolytic cleavage of fatty acyl hydroperoxides. Here, •O2- radicals are formed as by-products of lipid peroxidation (reaction 13, 14). Fe3+ + ROOH → Fe3+ - HO• + RO• (13) Fe3+ + HO• + ROOH → Fe3+ H2O + ROO• (14) The present study obtained comprehensive results on the redox state of Walker-256 carcinosarcoma showing that all of the proposed treatment modalities caused an increase in the level of free iron, lactoferrin and NO-FeS-proteins. Another clinically relevant finding was that the value of the growth factor φ (Table 1) sought to evaluate tumor growth kinetics depended on the parameters of a spatially inhomogeneous magnetic field (Fig. 2). Therefore, these investigations provided further evidence for MN treatment in combination with the local inhomogeneous CMF to modulate iron-catalyzed Haber-Weiss cycle and iron-dependent lipid peroxidation as a mechanism for free radical formation in


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Walker-256 carcinosarcoma, eventually, leading to cancer cell damage, necrosis and apoptosis. Nonlinear phenomena in magnetism have been widely investigated to describe chaotic dynamics of anisotropic magnetic NP in the presence of an external magnetic field. The magnetization of NP rather follows a diverse and complex behavior such as bistability, quasiperiodicity and chaos.38 The evidence from this study concluded that the inhomogeneous CMF with different strengths applied to heterogeneous tumor tissues induced different magnetic anisotropy in MN which had a significant influence on nonlinear kinetics, redox state and histological pattern of the tumor. Since cancer cells have a more heterogeneous structure than normal cells, the entropy of cancer cells is different from normal ones. The reversal of the entropy flow between cancer and normal cells can result in reversed signal transmission and so tumor regression.39 Consequently, the magnetic anisotropy of MN could have switched the behavior of redox reactions between a chaotic (oscillating) and ordered (regular) manner under non-equilibrium conditions in the tumor and microenvironment.40 In terms of Darwinian dynamics that occurs during somatic evolution, growing tumors form recognizable architectural features


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with high spatial and temporal heterogeneity. As reported above (Fig. 5), a statistical measure of tumor heterogeneity revealed that the minimum value of the entropy was found in group 3 and 4. The lowest value of the growth factor φ and the highest survival rate tendency (70% and 40%) were observed in animals assigned to the 3rd and 4th groups 90 days after tumor implantation. These results are consistent with previous studies of intratumor heterogeneity and have linked tumor heterogeneity with treatment response, tumor resistance and disease progression.41, 42 Experimental Models and Methods

Magneto-mechano-chemical synthesis

The high-precision magneto-mechanical milling chamber (NCI) was loaded with iron oxide (Fe3O4) NP (30-50 nm particle size, Sigma-Aldrich) and the antitumor antibiotic DOXO (Pfizer, Italy). During a five-minute MMCS the chamber was vibrated at a frequency of 36 Hz with an amplitude of 9 mm, and energy of 20 W/g. MN were exposed to electromagnetic fields with a frequency of 42 MHz, initial power of 75 W and CMF of magnitude B = 12 mT for MMCS. MN was defined as having a mass ratio of 1:2 for DOXO


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and NP with a zeta potential of + 32 ± 2 mV measured by Malvern Zetasizer and hydrodynamic size of 108 ± 19 nm. The MN loading was held fixed at 33%. Parameters of MMCS were chosen according to earlier design phase investigations on efficacy, loading, release and stability of MN in a physiological saline medium. In vitro tests of NP cytotoxicity on human liver cancer cells HepG2 have shown little or no toxicity to hepatocarcinoma cells. A complete description of this study is given in our previous paper43. The magnetic properties and ESR spectrum characteristics of MMCS for MN and DOXO are highlighted in Table 2. Table 2. The magnetic properties and electron spin resonance spectrum characteristics of MN after magneto-mechano-chemical synthesis

Sample

g-factor

Saturation

Coercive

Area of the

magnetic

field Hc, Oe

hysteresis

signal

loop, erg/g

intensity,

moment ms, emu/g

Conventional DOXO

—*

ESR

r. u. —

—

2.003

0.027

—

—

2.003

0.033

(without

activation) Magneto-

—†

mechano-


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chemically treated DOXO MN *Conventional

11.81

12.57

3402.0

2.37

1.00

DOXO (without activation) is diamagnetic (m = – 0.200 emu/g) at 3 kOe;

†Magneto-mechano-chemically

treated DOXO was paramagnetic, the magnetic

moment

m = –1.18 emu/g at 3 kOe;

Magnetic-dipole applicator

MDA utilized an array of needle-shaped dipole antennas and neodymium permanent magnets with different magnetic field strength (Fig. 7).


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Figure 7. Magnetic-dipole applicator for local treatment of the tumor: 1 – holder; 2 – magnetic flux dipoles (diameter 0.5 mm, magnet pole separation 3 mm); 3 – dielectric; 4 – permanent magnet.

The horizontal slice of CMF for the magnetic dipole applicator (Fig. 8) was calculated considering the equations of magnetostatics44 and MAGNITHERM 1.0 software (NCI).


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Figure 8. The horizontal slice of CMF for the magnetic dipole applicator at a distance of 2 mm above the dipoles: (a) magnetic-dipole applicator (MDA 1) carrying a permanent magnet with the maximum magnetic field induction Bmax = 40 mT and adhesive force of 15 kg , (b) magnetic-dipole applicator (MDA 2) carrying a permanent magnet with the maximum magnetic field induction Bmax = 400 mT and adhesive force of 20 kg, (c) magnetic-dipole applicator (MDA 3) carrying a permanent magnet with the maximum magnetic field induction Bmax = 600 mT and adhesive force of 40 kg.

A 40 mg powder of Fe3O4 particles with the diameter of 30-50 μm (International Center for Electron Beam Technologies of E.O. Paton Electric Welding Institute, Ukraine) was used in an effort to visualize particle alignment by CMF on a dielectric surface with an area of 1590 mm2. Computer-aided image analysis (Table 3) revealed that the lowest


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average brightness level and the highest value of the entropy parameter was found in photographs of particles aligned by CMF with the MDA 2 applicator (Fig. 9).

Figure 9. Photographic visualization of particles aligned by CMF with the magnetic dipole applicator: (a) MDA 1, (b) MDA 2, (c) MDA 3.

Table 3. Computer-aided analysis of digital images of particle alignment by CMF with the magnetic dipole applicators

Magnetic-dipole applicator

Average brightness, arb. units

Entropy (E), arb. units

MDA 1

134.0

4.80

MDA 2

122.4

4.91

MDA 3

143.0

4.74


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Laboratory animals, tumor implantation and treatment of Walker-256 carcinosarcoma

Animal experiments were carried out using a strain of noninbred male rats weighing 135 ± 10 g bred at the vivarium of National Cancer Institute (Kyiv, Ukraine). Walker-256 carcinosarcoma cells (0.4 ml 20% cell suspension in the medium 199) were inoculated intramuscularly into the right thigh of rats. These animals were randomly assigned to the various groups (n = 12 per group): group 1 – control (no treatment); group 2 – administration of conventional DOXO only; group 3 – administration of MN + MDA 1; group 4 – administration of MN + MDA 2; group 5 – administration of MN + MDA 3. Treatment was performed 3 days after tumor inoculation. Rats were administered a single intravenous injection every other day for 5 times of either DOXO at 1.5 mg/kg body weight in 0.3 ml saline or MN comprising DOXO at 1.5 mg/kg body weight and NP at 3 mg/kg body weight in 0.3 ml saline. Protocols involving animals conformed to the Law of Ukraine N 3447–ІV on the protection of animals from cruelty and the European Directive 2010/63/EU on the


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protection of animals for scientific purposes. All procedures were approved by the Regional Committee for Animals and Medical Research Ethics of the National Cancer Institute.

Electron spin resonance spectroscopy

ESR spectroscopy is a technique to directly detect compounds with unpaired electrons. ESR measurements were performed with a RE1307 spectrometer at liquid nitrogen temperatures (77 K) using a cylinder resonator in the H011 mode with a frequency of 9.15 GHz. ESR parameters were as follows: microwave power 40 mW and modulation frequency 100 kHz. The samples were inserted into the quartz Dewar with an inner diameter of 4.5 mm. Once the spectra from samples were recorded, the g-factor value and concentration of paramagnetic centers were calculated. The equation of the resonance condition was used to calculate the g-factor: hv = gβB, (15) where hv = 9.15 GHz is the microwave frequency, β = 1.39968 GHz/kGs is the Bohr magneton, and B is the magnetic induction.


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ESR spectra were described as:45,

46

free iron (g = 2.2–2.4); ubisemiquinone (g =

2.0023); ESR signal (g = 2.007) with a triplet structure was recorded in the semiquinone radicals for iron-sulfur (NO-FeS)-proteins of N-type complexes in mitochondria and the accumulation of multifunctional protein lactoferrin (g = 4.25).

Analysis of nonlinear kinetics of tumor volume

The assessment of the tumor growth kinetics was based on the growth factor  according to the autocatalytic equation.47 The braking ration  was used to evaluate the nonlinear kinetics of tumor growth in groups given the anticancer magnetic nanotherapy:  = c/e, (16) where c is the growth factor for the control and e is the growth factor for the investigated group.

Histopathologic examination of the tumor

H&E stains of formalin-fixed tissues were used routinely to determine tumor type. The collected tissue specimens were fixed in 10% neutral-buffered formalin (pH = 7.4). Tissue


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sections were removed from formalin and processed into paraffin blocks with a microwave histoprocessor Histos-5 (Milestone, Italy). Microm HM325 (ThermoScientific, Germany) to cut 5 μm thick sections. Tissue studies employed OLYMPUSGХ 41 microscope with QuickPHOTO MICRO 2.3 software to investigate and take digital images of the visualized tumor specimens under the standard conditions. The structural heterogeneity in specimens collected from muscle and tumor tissues was calculated by the entropy associated with each brightness level of digital photography. CHAOS & IMAGE 1.0 software (NCI) was exploited to analyze images. Statistical analysis

Data comparisons were performed with Statistica 13.0 (© StatSoft, Inc., 2015) software and statistical significance was analyzed using the Student t-test when the data followed a standard normal distribution. The Kolmogorov-Smirnov test determined whether data were normally distributed. The p-value was calculated as for a two-tailed test. Kaplan-


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Meier method was used for evaluation of survival. The survival data for animal groups were compared statistically with the log-rank test (p < 0.01).

Conclusion and Future Work

In summary, this paper provided additional evidence for modulation of the nonlinear magnetochemical effects during nanotherapy of Walker-256 carcinosarcoma based on the magneto-mechano-chemically synthesized MN comprising magnetite NP and DOXO under the local influence of a spatially inhomogeneous CMF upon iron-dependent redox reactions. The present findings contribute to the field of magnetochemistry which is a multidisciplinary platform for future implementations of cancer magnetic nanotherapy in oncology clinics through translational medicine.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript and contributed equally.

DECLARATIONS OF INTEREST: Authors have no conflict of interest to declare.


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ACKNOWLEDGMENT

This work was supported by the Programs of National Cancer Institute, Ukraine, Initiative Research BN.14.01.07.170-16.

ABBREVIATIONS MN, Magnetic nanodot; ROS, Reactive oxygen species; RNS, Reactive nitrogen species; RONS, Reactive oxygen and nitrogen species; CMF, Constant magnetic field; DOXO, Doxorubicin; MDA, Magnetic-dipole applicator; H&E, Hematoxylin and eosin; ESR, Electron spin resonance; NP, Nanoparticles; MMCS, Magneto-mechano-chemical synthesis;

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Figure 1. The growth kinetics of Walker-256 carcinosarcoma (M ± m): 1– control (no treatment); 2 – conventional DOXO; 3 – MN + MDA 1; 4 – MN + MDA 2; 5– MN + MDA 3. 83x41mm (600 x 600 DPI)



Figure 2. Kaplan-Meier curves displaying survival rates of animals on the 90th day after Walker-256 carcinosarcoma implantation: 1 – сontrol (no treatment); 2 – DOXO; 3 – MN + MDA 1; 4 – MN + MDA 2; 5 MN + MDA 3. All survived: *P < 0.01; versus сontrol (no treatment); using the log-rank test. 83x41mm (600 x 600 DPI)


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Figure 3. Histological sections of Walker-256 carcinosarcoma (H&E × 100) and the image entropy (E, arb. units): 1– control (no treatment) (E = 5.36); 2 – conventional DOXO (E = 5.30); 3 – MN + MDA 1 (E = 5.14); 4 – MN + MDA 2 (E = 5.14); 5 – MN + MDA 3 (E = 5.27); 6 – MN + MDA 2 (the muscle) (E = 4.85). 177x89mm (300 x 300 DPI)



Figure 4. Electron spin resonance spectra of Walker-256 carcinosarcoma on the 21st day after tumor implantation. 1 – control (no treatment); 2 – сonventional DOXO; 3 – MN + MDA 1; 4 – MN + MDA 2; 5 – MN + MDA 3; 6 – MN + MDA 2 (the muscle). Т = 77 К. 83x103mm (600 x 600 DPI)


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Figure 5. The redox state in Walker-256 carcinosarcoma on 19th day after tumor implantation.* Statistically significant difference from control group, p < 0.05;+ Statistically significant difference from conventional DOXO, p < 0.05;# Statistically significant difference from MN + MDA 1, p < 0.05;& Statistically significant difference from MN + MDA 2, p < 0.05;● Statistically significant difference from MN + MDA 3, p < 0.05; 174x79mm (600 x 600 DPI)



Figure 6. A suggested mechanism to explain magnetochemical effects of MN and a local nonlinear CMF on the inner mitochondrial membrane in malignant tumor cells: cellular redox potential (A), magnetic properties and conductivity (B), electrochemical gradient (C), electron and proton spin-dependent transport regulation leading to oxidative stress (D). 175x150mm (300 x 300 DPI)


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Figure 7. Magnetic-dipole applicator for local treatment of the tumor: 1 – holder; 2 – magnetic flux dipoles (diameter 0.5 mm, magnet pole separation 3 mm); 3 – dielectric; 4 – permanent magnet. 84x102mm (600 x 600 DPI)



Figure 8. The horizontal slice of CMF for the magnetic dipole applicator at a distance of 2 mm above the dipoles: (a) magnetic-dipole applicator (MDA 1) carrying a permanent magnet with the maximum magnetic field induction Bmax = 40 mT and adhesive force of 15 kg , (b) magnetic-dipole applicator (MDA 2) carrying a permanent magnet with the maximum magnetic field induction Bmax = 400 mT and adhesive force of 20 kg, (c) magnetic-dipole applicator (MDA 3) carrying a permanent magnet with the maximum magnetic field induction Bmax = 600 mT and adhesive force of 40 kg. 177x43mm (600 x 600 DPI)


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Figure 9. Photographic visualization of particles aligned by CMF with the magnetic dipole applicator: (a) MDA 1, (b) MDA 2, (c) MDA 3. 159x57mm (300 x 300 DPI)



Graphical Table of Contents 79x43mm (300 x 300 DPI)


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Nonlinear Magnetochemical Effects in Nanotherapy of Walker-256

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