Enhancement of RF-EMF Therapy by Means Selective Accumulation of

Glucose Conjugated Magnetic Nanoparticles in vivo in Rats

Traikov1 L., Antonov1 I.,Vesselinova2 L., Hadjiolova3 R., Raynov4 J.

1-Medical University-Sofia; Faculty of Medicine; Dept. Medical Physics and Biophysics

2- Military Medical Academy, MHAT Sofia, MMDER/Clinic of Physical and Rehabilitation Medicine

3-Medical University-Sofia; Faculty of Medicine; Dept. Pathophysiology

4- Military Medical Academy, MHAT Sofia, Clinic of Hematology

INTRODUCTION

Fe-Magnetic nanoparticles (Fe-MNP) have gained a lot of attention in biomedical and

industrial applications due to their biocompatibility, easy of surface modification and

paramagnetic properties.

The basic idea of our study is whether it is possible to use glucose-conjugate Fe-

MNP(Glc-Fe-MNP) for targeting and more accurate focusing in order to increase the effect of

high-frequency electromagnetic fields induced hyperthermia in solid tumors. Tumors

demonstrate high metabolic activity for glucose in comparison with other somatic cells.

Increasing of accumulation of Glc-Fe-MNP on tumor site and precision of RF-EMF

energy absorption in solid tumors, precede RF-EMF induced hyperthermia. Rat model for monitoring the early development of breast cancer. Twenty female

Wistar rats (MU-line-6171) were divided into two groups of ten rats that were either treated with N-methyl-N-nitrosourea to induce breast cancer and 10 with carrageenan to induce

inflammation (control). Glc-Fe-MNP can offer a solution to increase hyperthermia effect to the desired areas

in the body by accumulation and increasing local concentration due to high tissue metabolic assimilation.

In this condition, it is considered that the magnetization of the nanoparticles is a

single-giant magnetic moment, the sum of all the individual magnetic moments and is

proportional to the concentration of Glc-Fe-MNP.

There are several problems associated with the use of nanoparticles in cancer

treatment: (a) the need to create high local magnetic fields and appropriate thermal gradients

for the treatment; (b) temperatures in a range of 43–70 °C can be accompanied by the

formation of thermal resistance at the cellular level as a result of the initiation of heat shock

proteins synthesis; (c) the temperature above 45°C may shut down tissue perfusion, and can

change blood vessels permeability (see Fig.1.) and may require durations of 30–60 min,

placing strict and challenging technical requirements; (d) due to inhomogeneous magnetic

nanoparticles distribution, complete tumor eradication was not possible; (e) further-more, targeted radio frequency therapy with magnetic nanoparticles is often not suitable for

disseminated and abdominal tumors; (f) slow biodegradation of nanoparticles in the body and side effects of their accumulation in the liver, spleen, muscles, and other organs; (g) the

application of electromagnetic hyperthermia is usually very expensive due to high equipment and treatment costs Giustini,A.J. et al. 2010

Fig.1. Thermal Induced damage of blood vessels (L.Traikov, 2014)

To overcome mentioned above problems we developed technology of magnetic nanotherapy which utilizes magnetic nanoparticles with glucose conjugate to their surface

(Glc-Fe-MNP), focused from one side by heist metabolic rate of the tumor and from other side by a strong external inhomogeneous magnetic field to the tumor region. In this

technology of magnetic nanotherapy the antitumor magneto-thermal effect is initiated by free

radical reactions, due to overloading of redox-chains and extra production of reactive oxygen

substances (ROS), under mild hyperthermia at 37–39 °C. The magnetic fields effect observed

with radical pair recombination is one of the well-known mechanisms of magnetic fields

interaction with biological systems.

Exposure to magnetic fields can increase the activity, concentration, and life time of

paramagnetic centers (free radicals as a ROS), which might cause oxidative stress, and/or

apoptosis of tumor cells according, Orel,V.E. et al. 2013.

Though the standard therapies based on surgery, chemotherapy, irradiation, or

combinations of them steadily improve there are many attempts with a multitude of

alternative therapy concepts among which different types of hyperthermia have already

entered into clinical practice (Falk and Issels 2001).

In comparison with whole body hyperthermia where the systemic temperature (by means of a heat bath) has to be carefully controlled to, as 41.8 oC (Robins et al 1997), there

are different ways of effecting local intracorporal heat generation by means of microwave radiation, by capacitive or inductive coupling of radiofrequency fields, by implanted

electrodes, by ultrasound, or by lasers. Alternatively, in magnetically mediated hyperthermia one deposits magnetic material in the tumour which is heated by means of an external

alternating magnetic field. In comparison with the application of macroscopic magnetic implants (‘macro-

particles’) which are currently in clinical use for special cancer types, recent studies are

focused on magnetic nanoparticles as their heat generation potential appears beneficial and

they provide the opportunity of direct tumour targeting through blood circulation (Moroz et al

2002).

Two therapy modalities are commonly differentiated: treatments at temperatures of

42–45 oC for up to few hours—actually denoted as hyperthermia—need a combination with

other assisting toxic agents (mostly irradiation or chemotherapy) for reliable damage of

tumour cells.

In the following, both therapy modalities are comprised as magnetic particle

hyperthermia (MPH). In any case, the amount of nanoparticles to be applied should be as

small as possible. In order to reach the therapy temperature with minimum particle

concentration in tissue the specific heating power of the magnetic nanoparticles in magnetic

RF fields should be as high as possible. There is a multitude of known magnetic materials

which, however, for biomedical applications is strongly restricted by the demand of

biocompatibility.

Magnetic losses to be utilized for heating arise due to different processes of

magnetization reversal in systems of magnetic nanoparticles which depend in different

manners on the applied EMF amplitude and frequency. Moreover, there is a strong

dependence of magnetic particle properties on structural ones as a size.

In early research on magnetic particle hyperthermia either relatively large multidomain

particles (>100 nm) according Gilchrist et al 1957 or comparatively small superparamagnetic

particles (<10 nm), according Jordan et al 1999, were used. • We followed several tasks at current study: Synthesizing of Glc-Fe-MNP;

Characterization of Glc-Fe-MNP; Rat Skin-flap model of breast cancer; Infusion and

visualization; Exposure with RF EMF and hyperthermia.

The basic idea of our study is to investigates possibilities to use glucose-conjugate Fe-

MNP(Glc-Fe-MNP) for targeting and more accurate focusing in order to increase the effect of

high-frequency electromagnetic fields induced hyperthermia in solid tumors. Tumors

demonstrate high metabolic activity for glucose in comparison with other somatic cells.

MATERIALS AND METHODS

In present work we used modified method of E. Nakasone et al. 2013 Microscopes and

Imaging Software; Microscope- Carl Zeis fluorescent microscope systems and software are

used for the imaging of live unconscious rats. Fluorescence microscopy with 3CCD camera (Carl Zeiss AxioCam ICc1)

Image analysis. We use Origin Pro 7.0 (Origin Lab Corp. USA) and free image analyzer Image J (National Institute of Health [NIH]) for image analysis.

(Pre)-treatment of Animals for Imaging Rat model for monitoring the early development of breast cancer.

20 female Wistar rats (MU-line-6371) were divided into two groups of ten rats (n=10) that were either treated with: -N-methyl-N-nitrosourea to induce breast cancer (n=10) -

Carrageenan to induce inflammation (control). (n=10) . Inguinal mammary tumors are optimal for imaging using our protocol when the longest diameter measures approximately 8

mm or less by caliper measurement.

Fluorescein isothiocyanate-labeled dextran 250 kDa (FITC-dextran-250, 2.5 % (w/v)

in PBS, 50µl/25g body weight) was injected into the caudal vein to visualize vasculature of

Control and tumor induced rats.

Common ferrofluids due to a broad size distribution are rather flat, the gained fractions

exhibit clear peaks which could be shown by Algorithm for particle size determination by

means laser light scattering-680 nm, the largest diameter fraction (about 18 nm).

RESULTS

The original work must be substantially complete. Abstracts that do not specifically refer

to new results will not be considered for oral presentation. In order to followed blood vessels leakage in Control and Experimental conditions we

infuse FITC-Dex 250 kDa, for 10 min.

Normal blood vessels (range between 10 and 100 µm) has no permeable for FITC-Dex-

250 and integral curve of image densitometry at region of interest (ROI) show narrow curve

of fluorescent intensity. Leakage of cancer blood vessels (range between 10 and 30 µm) was

followed in Cancer-ROI by means FITC-Dex-250 as reference of FITC-Albumin.

The reference range for albumin concentrations in blood plasma is approximately 35 -

50 g/L (3.5 - 5.0 g/dL), with molecular mass of 66.5 kDa. By means extravasation of FITC-

Dex-250 kDa integrated fluorescence curve of ROI-cancer, is wider than the Control.

Most tumor vessels have an irregular diameter and an abnormal branching pattern and

do not fit well into the usual classification of arterioles, capillaries, or venules. Even large-

diameter vessels have thin, leaky walls.

Although some reports suggest that tumor vessels lack endothelial cells, pericytes

(mural cells), or basement membrane, recent work indicates that all of these components are

present but abnormal. Endothelial cells, although present on most if not all tumor vessels, do

not form a normal monolayer and thus do not have a normal barrier function. The cells are

disorganized and irregularly shaped. Endothelial cells of some tumor vessels overlap one

another, have luminal projections. The cells also have loose interconnections and focal

intercellular openings, which are likely to be responsible for much of the vessel leakiness.

(Donald M. et al 2002).

At visual light we observe process of Glc-Fe-MNP distribution by means

Histochemical visualization. Places of accumulation of nanoparticles correspond with places

nearby to blood vessel wall, extra-luminal space. Accumulation is time dependence process

and strongly depends by Leakage of the vessel wall therefore huge amount of Glc-Fe-MN

particles can be observed in tumor tissue nearby to blood vessel wall after 40 min after

infusion through caudal vein.

Fig.2. Visualization of Glc-Fe-MNP in skin flap model of breast cancer cells by modified Lilli

method, x100, oil immersion. a — Several Glc-Fe-MNP found in cells cytoplasm (Control). b

—cells with separate and enlarged granules of Glc-Fe-MNP. c —cells with big number of

Glc-Fe-MNP nanogranules, many small dense granules. d —cells in necrobiotic death due to

overaccumulation of large dense granules of Glc-Fe-MNP

CONCLUSIONS

Glc-Fe-MNP can offer a solution to increase hyperthermia effect to the desired areas

in the body by accumulation and increasing local concentration due to high tissue metabolic

assimilation.

In this condition, it is considered that the magnetization of the nanoparticles is a

single-giant magnetic moment, the sum of all the individual magnetic moments and is

proportional to the concentration of Glc-Fe-MNP.

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