*2.5. Release Study of Quercetin under Stationary and Alternating Magnetic Fields*

The apparatus setup scheme shown in Figure 2 enables the fine-tuning of the quercetinrelease kinetic profile under applied combined stationary and alternating magnetic fields. The magnetic field enforced in the experiment is a combination of stationary and alternating magnetic fields. The position of the permanent magnet fixed and stable towards the fixed magnetic coil placed within the reactor ensures the permanent and alternating magnetic fields perpendicular to each other [35]. Without going into more detail, we showed in previous work [35] that, by applying external magnetic fields, MNPs behave as Brownian particles with quasi-periodic movement that enables loaded drug molecules to became enhanced released from the MNPs. A magnetic field is an effective stimulus with deep penetration capacity. The high-frequency alternating magnetic field (HF-AMF), from 50 to 400 kHz, and low-frequency alternating magnetic field (LF-AMF), from 0.1 to 5 kHz, have been employed to trigger the release of drugs from nanocarriers [36]. In our previous work, we employed the LF-AMF to induce the flavonoid release from the magnetic aggregates [35]. To get more insights into the effect of the combination of applied permanent and AMF to drug release, we designed sophisticated HF-AMF instrument to induce increased drug release at HF-AMF having in mind Brezovitch criterion [37]. He proposed a safety limit

where the product of magnetic field and amplitude frequency (H0×f) should not exceed 4.85 × 108 A m−<sup>1</sup> <sup>s</sup>−<sup>1</sup> to avoid any harmful effect on the organism. In our study, the product amounts 4 mA m−<sup>1</sup> s−1, 1 mA m−<sup>1</sup> s−<sup>1</sup> and 0.3 mA m−<sup>1</sup> for 100 kHz, 50 kHz and 10 kHz, respectively, indicating that we chose good frequencies and applied magnetic fields for any possible safe application to patients. In consideration of the design of our experiment, where there were relatively high frequencies of alternating magnetic fields (from 10 to 100 kHz), it was expected that the release of quercetin would be significantly enhanced by the influence of higher frequency.

**Figure 2.** Experimental setup of the release kinetics and schematic illustration of a reactor.

The drug release was calculated by measuring the absorbance of quercetin released in the supernatant at *λ* = 330 nm by the UV-Vis spectrophotometer. The releasing kinetics of quercetin from magnetite MNPs was investigated at three temperatures (25 ◦C, 30 ◦C and 37 ◦C) in the mixture PBS/EtOH (vol. 50/50) in which the solubility of quercetin totals to 5.66 mg/mL, 5.83 mg/mL and 6.02 mg/mL at 25 ◦C, 30 ◦C and 37 ◦C, respectively [38]. For the use of NPs as a drug delivery system, a physiological temperature of 37 ◦ C is of crucial importance. However, before the application of MNPs, they must be adequately stored. There are studies on the storage of MNPs at low temperatures, and the influence of low temperatures on the magnetic properties of MNPs [39]. Although the temperature effects on the magnetic properties of magnetite are very weak, MNPs contain a coating of organic material, in our case PEG 4000. Since PEG 4000 has crystallization temperatures at around 32 ◦C [40], which means that by changing the temperature of the MNPs suspension from the storage temperature to the ambient temperature, and further to the physiological temperature of 37 ◦C, the surface properties of MNPs and the relaxation kinetics could be changed. For this reason, measurements were made not just at 37 ◦C, but also at 25 and 30 ◦C to see how much the temperature change affects the drug-release kinetics. In addition, similar studies of the mechanism of release kinetics from NPs at different temperatures (10, 22 and 37 ◦C) have been conducted, for example, in study by Gronczewska [41], in that drug release and matrix degradation of polymer microspheres with different glass transition temperatures were investigated at various temperatures in order to clarify the effect of temperature on mechanisms of drug release. Then, 100 mg of quercetin-loaded MNPs were transferred in a dialysis bag (MWCO 8 kD, Thermo Fisher Scientific (Waltham, MA, USA)), after which 1 mL of PBS/EtOH (Vol. % = 50:50) medium was added and closed with dialysis bag clip holders. The dialysis bag was placed in a glass cylindrical reactor with a thermostatic jacket and flange size LF 100 containing 150 mL of PBS/EtOH mixture. The overhead stirrer (Ministar 20 control, IKA-Werke GmbH&Co (Staufen, Germany)) is going through the centre neck flat flange lid. The stirrer was set to 200 rpm. At selected time intervals, 1 mL of supernatant was replaced with fresh PBS/EtOH mixture through one angled side neck flat flange lid. The reactor is connected to a refrigerated–heating circulator (Corio CD-201F Julabo GmbH (Seelbach, Germany)) to control the appropriate temperature. The controlled release of quercetin was tested using appropriate external alternating (10 kHz, 50 kHz and 100 kHz) and stationary magnetic fields (*B* = 7.9 mT

and 11.0 mT) at controlled electric current (*I* = 100 mA). An external alternating magnetic field was achieved with a function generator (Wavetek 164 30 MhZ (San Diego, CA, USA) connected to the coil (N = 270, *l* = 4 cm). Experiments were performed using the magnetic field system set up from permanent disk-shaped magnets (rare earth) and solenoid with permalloy core connected to signal generator alternating 100 mA current. A reactor with the sample was placed among the two magnetic fields. Defining the *O*xy plane as the surface of the liquid, the permanent field was along the *O*<sup>z</sup> axis and the alternating field along the *O*<sup>x</sup> axis. Weak fields were applied in all experiments: the strength of the static permanent magnetic fields at the appropriate distance between the membrane dialysis bag and the permanent magnet was *B* = 7.9 mT and 11.0 mT. They were placed on a stand inside the reactor and used as sources of the permanent magnetic field. The function generator is connected to the oscilloscope (DS1000Z, Rigol Technologies (Beijing, China), which allowed the observation of the sinus waveform signal. The release kinetics of quercetin from magnetite MNPs was quantified by UV/VIS spectrophotometry (Photolab 7600 UV/VIS spectrophotometer Xylem (New York, NY, USA)). The linearity of the calibration was found to be valid from 1 × <sup>10</sup>−<sup>6</sup> mol dm−<sup>3</sup> to 1 × <sup>10</sup> <sup>−</sup><sup>4</sup> mol dm−<sup>3</sup> with correlation coefficients for quercetin all approaching 1.00. All release kinetics experiments have been performed in duplicate.

#### **3. Results and Discussion**
