Transmittance Measurements in Non-alternating Magnetic Field as Reliable Method for Determining of Heating Properties of Phosphate and Phosphonate Coated Fe3O4 Magnetic Nanoparticles

Different phosphates and phosphonates have shown excellent coating ability toward magnetic nanoparticles, improving their stability and biocompatibility which enables their biomedical application. The magnetic hyperthermia efficiency of phosphates (IDP and IHP) and phosphonates (MDP and HEDP) coated Fe3O4 magnetic nanoparticles (MNPs) were evaluated in an alternating magnetic field. For a deeper understanding of hyperthermia, the behavior of investigated MNPs in the non-alternating magnetic field was monitored by measuring the transparency of the sample. To investigate their theranostic potential coated Fe3O4-MNPs were radiolabeled with radionuclide 177Lu. Phosphate coated MNPs were radiolabeled in high radiolabeling yield (> 99%) while phosphonate coated MNPs reached maximum radiolabeling yield of 78%. Regardless lower radiolabeling yield both radiolabeled phosphonate MNPs may be further purified reaching radiochemical purity of more than 95%. In vitro stabile radiolabeled nanoparticles in saline and HSA were obtained. The high heating ability of phosphates and phosphonates coated MNPs as sine qua non for efficient in vivo hyperthermia treatment and satisfactory radiolabeling yield justifies their further research in order to develop new theranostic agents.


Introduction
In the past few decades magnetic nanoparticles (MNPs) have attracted far-reaching attention due to the expeditiously growing possibilities for their applications such as nanomaterial-based catalysis, biomedicine and tissue specific targeting, magnetic resonance imaging, magnetic particle imaging, data storage, environmental remediation, etc. [1,2]. Due to their extraordinary physico-chemical qualities, MNPs are especially used in the biomedicine. The MNPs may be similar to cell, virus, protein or gene size, but also they can easily enter a variety of cell structures. The heating of MNPs by changing the orientation of their magnetic domains in an alternating external magnetic field is substantial for various medical applications such as hyperthermia and triggered drug delivery to the targeted region of the body by induced self-heating [3,4]. However, surface functionalization of MNPs is pivotal for the fruitful application in medicine. Coating of MNPs increases the colloidal stability, prevents aggregation and agglomeration of the particles and provides nontoxicity in physiological conditions. It is noteworthy that phosphates and phosphonates are water soluble and biocompatible and allow convenient stability and reduced cytotoxicity of MNPs for medical application [5]. Furthermore, they have a large binding affinity to the bone tissue which allows application in various medical conditions such as osteoporosis and bone tumors [6,7]. In addition, phosphates and phosphonates are higly efficient chelators. Bisphosphonate-based coordination complexes with 99m Tc have been widely used for bone scintigraphy, due to their high sensitivity, specificity, and accuracy for detecting skeletal metastatic diseases [8] The binding of phosphates and phosphonates to the surface of MNPs and labeling with different radionuclides make them appropriate for combined radionuclide-hyperthermia therapy or SPECT/ PET-MRI diagnostics [5,9]. 177 Lu has become favored in recent years in the therapy of neuroendocrine and prostate tumors, due to very suitable physicochemical characteristics (T 1/2 = 6.7 days, E βmax of 497 keV (78.6%)) [10]. An additional advantage of using 177 Lu is γ-ray emission (E γ of 208 keV (11%) and 113 keV (6.6%)) during its decay which allows the monitoring of therapy. There are a small number of studies that refer to the potential use of 177 Lu labeled MNPs in cancer radionuclide therapy [11].
In the present study heating capacity of Fe 3 O 4 based MNPs coated with inositol hexaphosphate (IHP), imidodiphosphate (IDP), hydroxyethylidene diphosphonic acid (HEDP), and methanediylbis(phosphonic acid) (MDP) has been investigated for possible hyperthermia treatment. In order to analyze and explain the heating capacity of synthesized samples minutely, we monitored the behavior of MNPs in a non-alternating magnetic field by measuring laser transparency and correlated obtained data with hyperthermia measurements. Further, phosphate and phosphonate functionalized MNPs were labeleled with 177 Lu with the aim to investigate their potential for use in diagnosis or hyperthermia cancer therapy.
The excess of unreacted coating ligand was eliminated by dialysis against deionized water for one day.

Measurements in an Alternating and Non-alternating Magnetic Field
The magnetic hyperthermia efficiency of Fe 3 O 4 -MPD, Fe 3 O 4 -HEDP, Fe 3 O 4 -IDP, and Fe 3 O 4 -IHP MNPs was analyzed by using Commercial AC applicator (model DM100, nB nanoscale Biomagnetics). The heat generation under alternating magnetic field (30 mT) and the resonant frequency of 397 kHz was measured directly on the samples dispersed in water. The heating ability of MNPs (2 mg/ ml), defined as specific power absorption (SPA), calculated according to the following formula: SPA = (C p ·m w / m m )·(∆T/∆t), where Cp is the specific heat capacity of the medium (C p ~ C water = 4.18 Jg − 1 K − 1 ), m w and m m are the masses of the medium (water) and the magnetic nanoparticles, and ∆T/∆t is the initial slope of the time dependent temperature curve [14].
For the analysis of the sample in non-alternating external MF, device shaped in our laboratory was used [15]. Sanyo laser diode DL5147-040 in the single mode regime at wavelength λ = 655 nm was applied. Transmitted laser light was measured with a photodiode.

177
Lu-labeling of phosphates and phosphonates coated Fe 3 O 4 MNPs was obtained using the method previously described [11,16]. Briefly, 177 LuCl 3 solution (approximately 185 MBq in 5 µl) was added to an aqueous suspension of coated Fe 3 O 4 MNPs (5 mg/ml at pH 4.5) and incubated at room temperature with continual stirring for 1 h. To quantify the radiolabeling yield of 177 Lu-labeled MNPs and their radiochemical purity after purification by magnetic decantation, ITLC was performed on SG sheets with 0.1 M acetate buffer as the mobile phase. In this system, 177 Lu-labeled MNPs remained at the origin (Rf = 0.0-0.1), while the unbound 177 Lu 3+ migrated with the solvent front (Rf = 0.8-0.9).

In Vitro Stability of 177 Lu-MNPs
The in vitro stability of purified 177 Lu-labeled coated MNPs was determined in saline or human serum solution (total volume of 2 ml) by measuring the free, unbound 177 Lu in relation to the 177 Lu-labeled coated MNPs (bound 177 Lu) during incubation at 37 °C for 96 h. Small amounts of sample (50 µl) were taken at different time points (1,24,48, and 96 h) and analyzed by ITLC (SG plates) using 0.1 M acetate buffer as the mobile phase. In order to apply magnetic hyperthermia, it is necessary to take into account physiological limitations. Due to the eddy currents, MFs of high frequencies can cause local heating also in the sections of the tissue where no magnetic particles have been found. Along with clinical restrictions, technical issues should be also taken into account, since most studies on biological samples encompass a tight frequency range. The applied frequencies, together with the amplitude of the alternating field, are mainly based on literature data [17,18]. The temperature increase of Fe 3 O 4 -MPD, Fe 3 O 4 -HEDP, Fe 3 O 4 -IDP, and Fe 3 O 4 -IHP MNPs (5 mg/ml) as a function of the time was evaluated under a frequency of 397 kHz and magnetic field strength of 30 mT (Fig. 1).

Measurements in an Alternating and Non-alternating Magnetic Field
Although all specimens under investigation have shown substantial heating capacity, it is obvious that Fe 3 O 4 -HEDP MNPs achieved the highest temperature values. In order to explain obtained trends, we employed the analysis of MNPs behavior in a non-alternating magnetic field, by measuring laser transmittance [19][20][21][22][23]. Previously this method gave satisfactory valuable data [15,24]. The analysis was carried out at 30 mT and 400 mT and the results are depicted in Fig. 2. At the beginning of the measuring, the MF is switched off, and the specimens showed initial transparency. By employing the MF, the transparency of the samples abruptly decreases. The magnitude of this decrease depends on the specimen's type and field strength. Regardless of the field used Fe 3 O 4 -MPD and Fe 3 O 4 -HEDP MNPs showed a lesser decline of transparency in comparison to Fe 3 O 4 -IHP and Fe 3 O 4 -IDP MNPs. After some time (app 60 s) sudden increase of relative transmittance has been observed, due to the zippering of magnetic chains. It is noteworthy that external MF arranges MNPs along the field lines, like miniature magnetic needles [15,20,23]. Such ordering of magnetic domains causes strong mutual attraction and subsequent formation of magnetic chains. In addition, the  magnetic chains are arranged in space thereby building a quasi-lattice made of parallel lined magnetic threads. The source of laser is positioned in such a manner that light propagates through the quasi-lattice parallel with lines of non-alternating MF. Contrary to the case when nanoparticles are chaotically distributed, magnetic chains encounter a much lesser number of scattering centers. In other words, the quasi-lattice possesses far smaller cross section for scattering compared to randomly distributed particles [15,20,23]. As a consequence, the intensity of the transmitted light rises. The slope of the increase is very steep when the field of 400 mT was employed, whereas for the lower fields the increase is gradual. Previous study has shown that depth and width of the well depend on MF strength and kind of ferrofluid sample [15,25]. Comparing the results from Figs. 1 and 2 it is obvious that the heating capacity of the sample (Fig. 1.) stands in correlation with depth and width of the well (Fig. 2.). The specimens displaying the highest heating capacity in alternating MF, By comparing the amount of precipitate formed in the time regime when the field was operative (Fig. 3. samples  1b, 2b, 3b, 4b) and also at the saturation point (Fig. 3, sam-ples1c, 2c, 3c, 4c)  In addition to the practiced MF strength and frequency, other factors such as particle size and shape along with the concentration of the sample significantly influence the behavior of MNPs in non-alternating and alternating MF [26]. Generally, more concentrated dispersions of MNPs broaden the application, therefore various electric and magnetic fields can be used. By diminishing the concentration of the sample, SPA values decline, and below 2 mg/ml heating effect could not be detected (Fig. 4).
The behavior of Fe 3 O 4 -HEDP MNPs in non-alternating MF (400 mT) for different concentrations is depicted in Fig. 5. The field of 400 mT has been used in order to compare the well depth at different concentrations of Fe 3 O 4 -HEDP MNPs, since at 30 mT the wells are not distinct, and effect is very poor. At the highest tested concentration (8 mg/ml) the initial transparency, when the MF is switched off, is the lowest. By decreasing the concentration the initial transparency of the sample rises. The depth of the well increases with the dilution until the concentration of 1 mg/ml. This concentration for this particular sample is turning point, from which the decrease of the well depth starts (the effect is getting weaker). At the lowest concentrations (0.1 and 0.2 mg/ml), the well could not be observed and the increase of transparency is very small reaching only the initial value (when the MF was switched-off). Observations are in correlation with hyperthermia studies, i.e. the SPA values decrease with a dilution of the sample till the concentration  (Fig. 4.). Since the shape of the well depends on the magnetic properties and the applied concentration of the sample, by finding a turning point with a sharp decrease in the depth of the well, it is possible to detect the concentration at which there is a loss of heating capacity.

Radiolabeling and In Vitro Stability of 177 Lu-MNPs
The aim was to optimize the 177 Lu radiolabeling of coated MNPs toward their further in vivo applications. 177 Lu as trivalent metal usually easily make complexes at room temperature with MNPs that possess available phosphates and phosphonates functional groups on the surface. The radiolabeling yield of 177 Lu-MNPs as well as their radiochemical purity after purification by magnetic decantation was determined using ITLC-SG chromatography with 0.1 M acetate buffer as the mobile phase (

Conclusions
The results given, point out that the MDP, HEDP, IDP, and IHP-coated MNPs could achieve significant heating capacity at 397 kHz and 30 mT frequencies and thus show potential for biomedical applications. Based on the interaction of   The dilution of the sample leads to a lowering of heating capacity, and below 2 mg/cm 3 heating effect could not be detected. Additionally, substantial changes in the laser transmittance curve profile have been recorded below the concentration of 2 mg/cm 3 . Based on the present study continual measurement of laser transmittance through the MNPs samples in the non-alternating magnetic field has shown as a good method for assessment of MNPs heating capability. Furthermore, phosphate and phosphonate coated MNPs are chosen to be labeled with 177 Lu. Future studies should investigate the potential of 177 Lu-MNPs as theranostic agents, for diagnostic imaging and therapeutic hyperthermia as well.