J IRAN CHEM SOC (2015) 12:1553–1559 DOI 10.1007/s13738-015-0627-7
ORIGINAL PAPER
The effects of electric and magnetic fields on Lucifer Yellow fluorescence in electro and magnetopermeabilization studies Tahereh Pourmirmirjafari Firoozabadi1 · Azam Izadi2
Received: 11 October 2014 / Accepted: 6 March 2015 / Published online: 19 March 2015 © Iranian Chemical Society 2015
Abstract The effects of external electric and magnetic fields on Lucifer Yellow (LY) fluorescence spectrum have been studied. Fluorescence spectrum is quenched by external electric field in the order of 10–150 V/cm with frequency range 1–6000 Hz applied by the ECT-SBDC electric system. A significant increase in fluorescence spectrum peak was observed in our steady-state measurements while using high power external magnetic pulses of 0.6–3.2 Tesla strength with three different frequencies 0.25, 1, and 10 Hz applied by the TMS magnetic system. The purpose of selected protocols has been to examine the results of our studies on electroporation and magnetopermeabilization outcomes. In addition, the effects of electric and magnetic fields being simultaneously induced have been discussed. In the presence of a magnetic field, the effect of electric field on fluorescence is less effective. Keywords Electropermeabilization · Magnetopermeabilization · Lucifer Yellow · Quenching
Introduction Nowadays external electric and magnetic fields are used to permeablilize cell membrane with respect to drugs, ions, and macromolecules. Applying electric fields in electroporation, and magnetic fields in magnetopermeabilization, is
* Tahereh Pourmirmirjafari Firoozabadi
[email protected];
[email protected] 1
Department of Physics, Varamin‑Pishva Branch, Islamic Azad University, Varamin, Iran
2
Department of Physics, K. N. Toosi University of Technology, Tehran, Iran
a method of permeabilizing cell membrane. Cell membrane permeabilization under electroporation and magnetopermeabilization occurs when they are exposed to electric and magnetic pulses. Electrochemotherapy (ECT) is an efficient technique for patients who suffer from cancer. Historically there are some different protocols in electroporation. In the standard protocol, high electric field strengths (hundreds of V/ cm) and short duration pulses (µs–ms) [1–3] are used. In nano-electroporation, electric pulses with the strengths in the order of 0.3 MV/cm and nanosecond pulses are applied. One of the other approaches is using low electric field strengths in the order of 100 V/cm with high frequencies. As a result of this, an effective treatment in ECT by 4000 pulses and 4 kHz repetition frequency in low electric field amplitude has been reported by Shankayi et al. [4]. Using pulses with higher repetition frequency than the frequency of tetanic contraction or lower electric field strength leads to the reduced number of muscle contractions and associated unpleasant sensations during pulse delivery. There are various methods in electroporation and magnetopermeabilization to determine the amount of cell membrane permeabilization, using fluorescent markers is one of them. Due to eliminating the issues of working with radioactive tracers in the techniques (for most biomedical measurements), florescence spectroscopy has been generalized as a dominant procedure in a wide variety of studies. Not interacting with cell components and non-toxicity makes Lucifer Yellow (LY) as a quantitative detector in electroporation and magnetopermeabilization processes among other markers [5]. Any change in the LY spectrum is considered as an indication of cell membrane permeability [6–8]. Additionally, there are different experimental studies that demonstrate that fluorescence spectrum is affected by external electric [9] and magnetic [10] fields. According
13
1554
J IRAN CHEM SOC (2015) 12:1553–1559
to their steady-state measurements [11], external electric fields in the order of 100 kV/cm to MV/cm result in fluorescence quenching. In order to elucidate the mechanism of the electric field effect on each fluorescence components, picosecond time-resolved decay measurements were conducted [12]. Furthermore, synergy effects of electric and magnetic fields cause the electric-field-induced quenching to become smaller in the presence of magnetic fields [13]. Based on the fact that fluorescence spectrum is highly affected by external fields, studying LY spectrum under external fields, as the best candidate for biomedical studies specifically in the Electrochemotherapy, is the main objective of our study. In this study, the effect of low electric field strength and high power magnetic field on the LY fluorescence has been examined based on its applications in the electroporation and magnetopermeabilization protocols. Furthermore, we have studied the simultaneous effect of electric and magnetic fields on the fluorescence intensity under steady-state experiments. It should be noted, direct measurement of magnetic-field-induced fluorescence spectra can be informative in many disciplines specifically in the magnetopermeabilization studies. The reason that such low amplitudes of electric and magnetic fields were chosen was based on the fact that in the low electric field electroporation, the electric field amplitude is of the order 100 V/cm, and it was the specific intention to compare our results eventually with higher amplitudes. The reasoning behind this is that in nano-electroporation should the quenching effects be positive in low amplitudes then it will be much more likely with higher amplitudes; had we conducted the research with the higher amplitudes we could have not concluded that the quenching effects would be positive for the lower electric field amplitudes.
Material and methods Fluorescent dye To determine the quenching of fluorescence spectra, fluorescent dye LY (Sigma, St. Louis, MO) diluted in phosphate-buffered saline (PBS) with 0.25, 0.5, 1 and 1.5 mM concentration was used. All experiments were done in room temperature and dark laboratory. The fluorescence intensity
was measured offline in arbitrary units on a multiwall spectrofluorometer (Shimadzu RF-5000, Japan) 40 min after the exposure of the LY to electric and magnetic pulses. The excitation and emission wavelengths were set at 420 and 525 nm, respectively. The amount of florescence measured for control group not exposed to the pulses. Electric pulse exposure Electric pulses were applied to the fluorescence samples by an ECT-SBDC (designed and made in the Small Business Development Center and Electromagnetic Laboratory of the Medical Physics Department of Tarbiat Modares University). In the first part of the study, 4000 pulses, 100 µs duration, square-wave electric pulses of 100 V/cm amplitude with repetition frequency of 4 kHz were delivered to the fluorescence suspension at four different concentrations 0.25, 0.5, 1 and 1.5 mM. In the next step, with selected LY concentration, the effect of electric pulse amplitudes and frequencies on fluorescence quenching was investigated. For electric field intensity analysis, we used eight different electric fields (10–150 V/cm with 20 V/cm increment) with 4000 square wave electric pulses and 4 kHz repetition frequencies. After electric field chosen, several repetition frequencies 1, 200, 400, 600, 1000, 2000, 3000, 4000, 5000 and 6000 Hz have been studied in order to track the behavior of fluorescence spectra as a function of frequency. Magnetic pulse exposure A Magstim generator (Magstim Rapid, Magstim Company, Spring Gardens, Whit land, UK) was used as a magnetic stimulator. Such devices produce a very strong and short discharge current in a coil, which is usually used for transcranial magnetic stimulation [14]. In the case of timevarying current passing through the wires, the produced magnetic field is also time dependent. This strong pulsed magnetic field (PMF) induces an electric field of the order of tens of volts per centimeter in the space around the coil [6, 15]. It has been demonstrated that figure-of-eight coils allow for a more focused and greater peak electric field than simple round coils [16, 17]. Thus, the optimum coil for our experiments to deliver the most intense fields with larger decay time constant, a 70-mm figure-of-eight coil was chosen. The Petri dishes containing the LY were placed under
Table 1 Magnetic pulse specification in experimental groups Frequency (Hz)
Pulse number = time duration (s)
Pulse number = time duration (s)
Pulse number = time duration (s)
0.25 1
28 Pulses = 112 28 Pulses = 28
56 Pulses = 224 56 Pulses = 56
112 Pulses = 448 112 Pulses = 112
10
13
28 Pulses = 2.8
56 Pulses = 5.6
112 Pulses = 11.2
J IRAN CHEM SOC (2015) 12:1553–1559
the coil where two windings meet and attached to the coil to expose cells to the strongest possible PMF. The duration of pulses in all experiments was the same and the investigated parameters were the repetition frequencies (0.25, 1 and 10 Hz), the number of pulses in each train (112, 56 and 28 pulses) and energy transfer. The main frequency considered in this study was 0.25 Hz to have several minute exposure time before warming up the device and the coil and the maximum possible number of pulses was about 112. The number of pulses for each pulse repetition frequency was established half and a quarter of 112 (i.e., 56 and 28 pulses). All experiments were repeated at least three times on different days (Table 1).
1555
Fig. 1 Normal fluorescence intensity with respect to concentration of solution
Statistical analysis All results are presented in bar graphs. Vertical bars represent standard deviation of the mean. Statistical analyses were performed by means of SPSS for windows 16.0 . (SPSS Inc., Polar Engineering and Consulting). All data were tested for normality. One-way analysis of variance (ANOVA) followed by least significant difference was performed; after that, statistical differences analysis was accomplished by t test. The P values of
