Regulation of Electromagnetic Perceptive Gene Using Ferromagnetic Particles for the External Control of Calcium Ion Transport

Developing synthetic biological devices to allow the noninvasive control of cell fate and function, in vivo can potentially revolutionize the field of regenerative medicine. To address this unmet need, we designed an artificial biological “switch” that consists of two parts: (1) the electromagnetic perceptive gene (EPG) and (2) magnetic particles. Our group has recently cloned the EPG from the Kryptopterus bicirrhis (glass catfish). The EPG gene encodes a putative membrane-associated protein that responds to electromagnetic fields (EMFs). This gene’s primary mechanism of action is to raise the intracellular calcium levels or change in flux through EMF stimulation. Here, we developed a system for the remote regulation of [Ca2+]i (i.e., intracellular calcium ion concentration) using streptavidin-coated ferromagnetic particles (FMPs) under a magnetic field. The results demonstrated that the EPG-FMPs can be used as a molecular calcium switch to express target proteins. This technology has the potential for controlled gene expression, drug delivery, and drug developments.


Introduction
Various methods of expressing a target protein using external stimuli have been studied. It is possible to express the desired protein via plasmid transfection [1] or viral transduction [2] with a magnetic field [3,4] and other methods [5,6]. Target proteins have been quantitatively expressed by external stimuli such as the control of substrate (isopropyl β-d-1-thiogalactopyranoside; IPTG) [7], temperature [8], nutrients [9], oxygen [10], and growth factors [11]. However, these controls take advantage of allosteric regulators, temperature, and growth factors used for normal protein expression. These are not the methods of expressing target proteins by active control that can be turned on or off by an external magnetic field. Various methods have been developed to express proteins at a desired period of time, using external-stimulation techniques [6,[12][13][14][15][16]. The photostimulation method focuses on neuronal differentiation and proliferation wherein using optimal conditions of flash optical stimulation lead to a significant increase in the number of cell nuclei and neurons differentiated on nanostructures [17]. It has been suggested that plasmonic excitation of gold nanoparticles can be used to stimulate and monitor localized Ca 2+ ion signaling in neurons. Nanoparticle assisted localized optical stimulation (NALOS) serves to be a valuable addition to the existing repertoire of techniques that involve light dependent control of neuronal activity [18]. However, methods that involve optical

Characterization of Magnetic Particles
FMPs [33] and SMNPs [34] were characterized by both transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were obtained using a JEOL JEM-2100F, Tokyo, Japan. Samples were prepared by casting a drop of particle dispersion onto a carbon-coated copper grid. The room-temperature magnetization curves were obtained by a temperature magnetization curves were obtained by a Quantum Design PPMS-9 magnetomete, San Diego, CA, USA.

EPG Transfection
HEK293T cells were transfected with pcDNA3.1-EPG+GFP and Lipofectamine 2000 in a glass-bottom dish according to standard protocols. Transfected cells were incubated for 48-60 h at 37 • C, and GFP-expressing cells were confirmed by fluorescence microscopy.

Biotin Conjugated HEK293T EPG+GFP Cells
Briefly, 1 mL of 0.1 mM sulfo-NHS-biotin in PBS was applied to the HEK293T EPG+GFP cells and maintained for 30 min at 25 • C. NHS-activated biotins react efficiently with primary amine groups (-NH 2 ) available on the surface proteins of HEK293T cells, that leads to streptavidin-biotin conjugation chemistry. After the reaction, cells were carefully washed twice with the CIS and labelled with Fura-2-AM. Before calcium imaging, 10 µL streptavidin-coated FMPs (1% w/v, 1 × 10 9 /mL) and SMNPs (0.5% w/v, 1.5 × 10 13 /mL) were added to the HEK293T EPG+GFP for 10 min by using a silicon tubing system. Later, unbound magnetic particles were removed by washing.

Calcium Imaging in HEK293T EPG+GFP Cells
Calcium imaging was performed on the HEK293T cells transfected with pcDNA3.1-EPG+GFP using Lipofectamine 2000 Reagent (Life Technologies, Inc., Carlsbad, CA, USA). The cells were used for imaging at 48 h post-transfection, once the maximum transfection efficiency was observed at this time point. The cells were subsequently washed three times with the CIS. The cells were loaded with 1 µM Fura-2-AM for 45 min at 37 • C. Then, the transfected cells were washed three times with the CIS and Fura-2-AM in the cells was de-esterified for 30 min at 37 • C. Culture dishes were placed into customized imaging chambers. An inverted Olympus 1X71 (Olympus Corporation, Tokyo, Japan) microscope was used to measure the fluorescence intensity changes of Fura-2-AM. The signal intensity changes reflect changes in the intracellular calcium concentration in the cells due to magnetic stimulation. Subsequently, GFP-positive cells were randomly selected using Metafluor imaging software (Molecular Devices, San Jose, CA, USA) and changes in the [Ca 2+ ] i were recorded as ratiometric alterations in both 340 and 380 nm wavelengths. A magnetic stimulus was applied by using the FMPs or SMNPs that subsequently showed magnetic field strengths of~200 and 0 µT at 100 s, respectively. The data were analyzed as changes in the ratiometric fluorescence intensity of Fura-2-AM over time, post magnetic stimulation.

Cell Identification
To identify responding cells (Responded cell; R), the fluorescence intensity of each individual cell was measured at 100 s prior to magnetic stimulation (I before ). The maximum fluorescence intensity of an individually responding cell was also measured after magnet particle stimulation (IMafter). The mean value (100-103 s) of I before was the I mean .The mean value (200-223 s) of IM after is IM mean and the standard deviation before magnetic stimulation is abbreviated SD. The measured responded cells were described as a numeric value at a given time point. Responding cells were determined using the following equation (Equation (1)) [32]: R = IM mean > I mean + (5 × SD). (1)

High-Resolution Fluorescent Microscopy Images
High-resolution fluorescent microscopy images of the cells were obtained using a Carl Zeiss LSM 710 laser-scanning microscope (Carl Zeiss, Oberkochen, Germany). HEK293T EPG+GFP cells were fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.5% Triton X 100 for 60 min. Post fixation, cells were dried and washed twice with PBS, followed by blocking with a 2% bovine serum albumin solution for 2 h. HEK293T EPG+GFP cells were stained with rabbit anti-EPG antibody (1:100 dilution) for 2 h followed by addition of donkey Alexa-595 conjugated anti-rabbit antibody (1:2000 dilution) with overnight incubation. Finally, a 1 mL (3nM) working solution of DAPI (4 ,6-diamidino-2-phenylindole) was added to stain the nuclei of the fixed cells, followed by mounting and sealing.

Statistical Analysis
GraphPad Prism software was used for statistical analysis and graphical representations of data. Student's t tests were performed on the data to evaluate significance. Non-significant values are shown as ns in the Results section, while *, **, and *** describe the p values of <0.05, <0.01, and <0.001, respectively.

Results and Discussion
The HEK293T cells were transfected with a pcDNA3.1 vector construct containing the EPG gene under the CMV promoter pcDNA3.1-EPG+GFP [32] ( Figure 1A). Figure 1B shows the schematic drawing of the biological circuit. The switch is turned on (irreversible) by magnetic particles (FMPs) that generate a local magnetic field around the target cells. The switch is turned on (irreversible) by magnetic particles (FMPs) that generate a local magnetic field around the target cells. This results in the activation of EPG thereby providing signal amplification. Calcium imaging confirms the current flow represented as light in the circuit. The final result is the expression of the target genes/proteins in cells represented as output in the circuit. To evaluate this biological circuit platform with the EPG, we designed a remote regulation of [Ca 2+ ] i in the HEK293T EPG+GFP cells. The amine functional groups of surface proteins on the HEK293T EPG+GFP cells were individually conjugated with biotin molecules by using sulfo-NHS-SS-biotin crosslinkers. Subsequently, streptavidin-coated magnetic particles were added at fixed time points during Ca imaging with Fura-2-AM. This setup allowed the monitoring of [Ca 2+ ] i with the addition of FMPs or superparamagnetic nanoparticles (SMNPs) that serve as an on/off switch for the expression of the EPG and [Ca 2+ ] i ( Figure 1C). The downstream pathways leading to subsequent increases in intracellular Ca is yet to be delineated. [Ca 2+ ]i imaging was employed to monitor the cell activation profiles with magnetic stimulation. EPG is a transmembrane protein; the origin of the Ca ion before the magnetic stimulation of cells is unknown; Ca ion is mainly stored in Endoplasmic reticulum (ER) but the uptake of Ca ion should be considered.

Characterization of Magnetic Particles
Transmission electron microscope (TEM) images confirmed the presence of FMPs and SMNPs (Supplementary Figure S1). The magnetic field strength of FMPs measured by the Gauss meter was 0.2 mT in 10 µL and was not detected in SMNPs (simply 10 µL of FMPs was dropped on the Gauss meter). This implies that the activation of the EPG was possible at as low as 0.2 mT by FMPs.

Expression of EPG
The HEK293T cells were transfected with pcDNA3.1-EPG+GFP and fluorescent imaging was performed after maximal expression was observed. Figure 2A show that the expression of EPG was localized to the cell membrane. Immunofluorescence staining was performed using primary anti-EPG antibodies and a secondary Alexa-595 anti-rabbit antibodies. This confirmed that the EPG was fluorescently stained red and was expressed in the cell membrane [32] (Figure 2A). Figure 2B shows that approximately 40% of HEK293T cells express EPG. [Ca 2+ ] i imaging was employed to monitor the cell activation profiles with magnetic stimulation. EPG is a transmembrane protein; the origin of the Ca ion before the magnetic stimulation of cells is unknown; Ca ion is mainly stored in Endoplasmic reticulum (ER) but the uptake of Ca ion should be considered.

Characterization of Magnetic Particles
Transmission electron microscope (TEM) images confirmed the presence of FMPs and SMNPs (Supplementary Figure S1). The magnetic field strength of FMPs measured by the Gauss meter was 0.2 mT in 10 µL and was not detected in SMNPs (simply 10 µL of FMPs was dropped on the Gauss meter). This implies that the activation of the EPG was possible at as low as 0.2 mT by FMPs.

Expression of EPG
The HEK293T cells were transfected with pcDNA3.1-EPG+GFP and fluorescent imaging was performed after maximal expression was observed. Figure 2A show that the expression of EPG was localized to the cell membrane. Immunofluorescence staining was performed using primary anti-EPG antibodies and a secondary Alexa-595 anti-rabbit antibodies. This confirmed that the EPG was fluorescently stained red and was expressed in the cell membrane [32] (Figure 2A). Figure 2B shows that approximately 40% of HEK293T cells express EPG.

Tagging EPG Expressing Cells with Magnetic Particles
In the previous study, we confirmed EPG activation by stimulation with either a 250 mT electromagnetic field (EMF) or a static magnet (25-50 mT) [32]. In this study we focused on fabricating an on/off switch that controls Ca 2+ ion channels using MNPs and the intracellular EPG expression and Ca response using a minimal local magnetic field (0.2 mT). GFP expression which is independent of EPG, due to its dual transcription with IRES was localized to the cell cytoplasm ( Figure 3A). It is important to note that the GFP is only indicative of the translation of the plasmid and not the localization of EPG.
Here, biotin was introduced with Sulfo-NHS-biotin to the amine functional group present on the cell surface [35] and fluorescence imaging was performed by binding Alexa 594-streptavidin. Cell surface binding of streptavidin to biotion was confirmed ( Figure 3A; in red). After Cy5-streptavidin-conjugated FMPs were added, the particles were uniformly distributed on the cell surface, as shown in Figure 3B. In addition, Cy5-streptavidin-conjugated SMNPs were similarly attached to the cells ( Figure 3C).

Tagging EPG Expressing Cells with Magnetic Particles
In the previous study, we confirmed EPG activation by stimulation with either a 250 mT electromagnetic field (EMF) or a static magnet (25-50 mT) [32]. In this study we focused on fabricating an on/off switch that controls Ca 2+ ion channels using MNPs and the intracellular EPG expression and Ca response using a minimal local magnetic field (0.2 mT). GFP expression which is independent of EPG, due to its dual transcription with IRES was localized to the cell cytoplasm ( Figure 3A). It is important to note that the GFP is only indicative of the translation of the plasmid and not the localization of EPG. Here, biotin was introduced with Sulfo-NHS-biotin to the amine functional group present on the cell surface [35] and fluorescence imaging was performed by binding Alexa 594streptavidin. Cell surface binding of streptavidin to biotion was confirmed ( Figure 3A; in red). After Cy5-streptavidin-conjugated FMPs were added, the particles were uniformly distributed on the cell surface, as shown in Figure 3B. In addition, Cy5-streptavidin-conjugated SMNPs were similarly attached to the cells ( Figure 3C).

Calcium Imaging
As shown in Figure 4, the signal intensity was increased when the FMPs were introduced to HEK293T EPG+GFP . However, when HEK293T GFP was treated with the SMNPs, the signal intensity did not change. This result suggests that different types of magnetic particles can be integrated as on or off calcium ion switches. Fura-2-AM is a ratiometric fluorescent dye that binds to free intracellular calcium. Once inside the cell, esterase enzymes cleave the AM groups, leaving Fura-2-free acid trapped inside the cell where it binds to Ca 2+ ions. It is excited at a ratio of 340/380 nm and emits fluorescence with a 510-nm peak [36,37]. Ca 2+ fluorescent signal intensity before and after magnetic

Calcium Imaging
As shown in Figure 4, the signal intensity was increased when the FMPs were introduced to HEK293T EPG+GFP . However, when HEK293T GFP was treated with the SMNPs, the signal intensity did not change. This result suggests that different types of magnetic particles can be integrated as on or off calcium ion switches. Fura-2-AM is a ratiometric fluorescent dye that binds to free intracellular calcium. Once inside the cell, esterase enzymes cleave the AM groups, leaving Fura-2-free acid trapped inside the cell where it binds to Ca 2+ ions. It is excited at a ratio of 340/380 nm and emits fluorescence with a 510-nm peak [36,37]. Ca 2+ fluorescent signal intensity before and after magnetic stimulation are shown for different conditions (Figure 4). When the SMNPs were added to the HEK293T EPG+GFP cellsfluorescent intensity was not changed significantly ( Figure 4A). When HEK293T GFP were stimulated with the FMPs, a change in fluorescence due to the 340/380 nm ratio was not observed ( Figure 4B). It implies that [Ca 2+ ] i was not changed with local magnetic stimulation due to the absence of the EPG in the cells. Microscopic images showed that the [Ca 2+ ] i of arbitrarily selected EPG+GFP positive cells significantly changed the 340/380 nm ratio due to FMP stimulation ( Figure 4C; red circles). Although not all of the HEK293T EPG+GFP cells responded to magnetic stimulation, the several cells showed that the concentration of the Ca 2+ ion was changed by FMP stimulation. This result confirms that the switch (magnetic particles) works and the transfection and function of the transistor (EPG) are successful.

FMPs Induce [Ca 2+ ]i Influx in Cells Expressing EPG
In Figure 4A, the HEK293T EPG+GFP + SMNPs showed a change in the 340/380 nm ratio of some cells. However, this was not significant. EPG-transfected cells activated an intracellular Ca ion increase that was significantl when assisted by selective magnetic particles. The HEK293T EPG+GFP cells were treated with 10 µL of FMPs or SMNPs at 100 s and the unbound particles were removed by a silicon tubing system. When the FNPs were applied to the HEK293T EPG+GFP , an initial decrease in the fluorescence intensity was observed for 20 s after a interval of 100 s ( Figure 5A). This time period is attributed to the absorption of light by the particles. A later increase in the fluorescence intensity (after 120 s) was due to the uptake of calcium into the cells. Unlike EPG-transduced neurons, response time is delayed in the HEK293T cells. This phenomenon should be studied further [32]. In Figure 5B, the reaction time of [Ca 2+ ]i was found to be between 120 s and 280 s following the addition of FMPs at 100 s. EPG-expressing cells were found to react within 180 s.
As a result, 40% ± 10% of the HEK293T EPG+GFP cells with FMPs responded to magnetic stimulation ( Figure 5C). In contrast, none of the HEK293T EPG+GFP cells with SMNPs or the HEK293T GFP cells with

FMPs Induce [Ca 2+ ] i Influx in Cells Expressing EPG
In Figure 4A, the HEK293T EPG+GFP + SMNPs showed a change in the 340/380 nm ratio of some cells. However, this was not significant. EPG-transfected cells activated an intracellular Ca ion increase that was significantl when assisted by selective magnetic particles. The HEK293T EPG+GFP cells were treated with 10 µL of FMPs or SMNPs at 100 s and the unbound particles were removed by a silicon tubing system. When the FNPs were applied to the HEK293T EPG+GFP , an initial decrease in the fluorescence intensity was observed for 20 s after a interval of 100 s ( Figure 5A). This time period is attributed to the absorption of light by the particles. A later increase in the fluorescence intensity (after 120 s) was due to the uptake of calcium into the cells. Unlike EPG-transduced neurons, response time is delayed in the HEK293T cells. This phenomenon should be studied further [32]. In Figure 5B, the reaction time of [Ca 2+ ] i was found to be between 120 s and 280 s following the addition of FMPs at 100 s. EPG-expressing cells were found to react within 180 s. The putative activation mechanism is represented in Figure 1. When a magnetic field is applied around the target cells, the magnetic particles promote the enhanced magnetic field around the target cells. Consequently, more EPGs are activated, leading to a local increase in the calcium concentration similar to the amplification of the calcium signal. However, further studies should carefully investigate whether the Ca 2+ influx is from an external or internal release from the calcium reservoir.

Conclusions
Cells were stimulated by a small magnetic field of less than ~0.2mT and EPG stimulation was confirmed to modulate Ca 2+ flux. The results of the present research confirm that the distance between the magnet and the cell is critical and further studies would verify the relationship. EPG-transfected cells activated an intracellular Ca 2+ ion increase by a magnetic field more significantly when assisted by selective magnetic particles. It was confirmed by calcium imaging using FURA2AM dye. Here we report for the first time, the construction of a biological circuit using a combination of magnetic nanoparticles and a genetically encoded protein (EPG) stimulated by a magnetic field. Together, the EPG and the FMPs create a selective calcium-signal-stimulating system (similar to a transistor in an electronic circuit) that can potentially trigger calcium influx and thereby activate calcium-sensitive promoters such as c-fos, BDNF, or NFAT and consequently selectively express target proteins for use in various biomedical applications such as cardiac and/or neuroscience research. It is confirmed that the EPG is related to the control of Ca ion channels but the exact pathway is unknown. The proper design of gene sequence could be the key to responsiveness to EMP. Further studies regarding possible mechanisms should be performed.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Characterization of magnetic particles, Figure S2 As a result, 40% ± 10% of the HEK293T EPG+GFP cells with FMPs responded to magnetic stimulation ( Figure 5C). In contrast, none of the HEK293T EPG+GFP cells with SMNPs or the HEK293T GFP cells with FMPs responded to magnetic stimulation ( Figure 5C). These results confirm that intracellular Ca ion increased by a magnetic field more significantly in EPG-expressed cells when assisted by selective magnetic particles. For a more accurate statistical analysis, we compared the 340/380 nm ratio intensities of 100 single cells before and after treatment. Changes in [Ca 2+ ] i were only observed in the HEK293T EPG+GFP cells ( Figure 5D, * p < 0.05, Supplementary Video S1) stimulated with FMPs, but not in the HEK293T EPG+GFP cells, stimulated with SMNPs or in the HEK293T GFP cells stimulated with FMPs ( Figure 5E,F, ns).
The magnetic particle cytotoxicity experiment confirmed that both SMNPs and FMPs were not cytotoxic at a concentration of 10 µL/mL that was used in the experiment (Supplementary Figure S2B).
The putative activation mechanism is represented in Figure 1. When a magnetic field is applied around the target cells, the magnetic particles promote the enhanced magnetic field around the target cells. Consequently, more EPGs are activated, leading to a local increase in the calcium concentration similar to the amplification of the calcium signal. However, further studies should carefully investigate whether the Ca 2+ influx is from an external or internal release from the calcium reservoir.

Conclusions
Cells were stimulated by a small magnetic field of less than~0.2mT and EPG stimulation was confirmed to modulate Ca 2+ flux. The results of the present research confirm that the distance between the magnet and the cell is critical and further studies would verify the relationship. EPG-transfected cells activated an intracellular Ca 2+ ion increase by a magnetic field more significantly when assisted by selective magnetic particles. It was confirmed by calcium imaging using FURA2AM dye. Here we report for the first time, the construction of a biological circuit using a combination of magnetic nanoparticles and a genetically encoded protein (EPG) stimulated by a magnetic field. Together, the EPG and the FMPs create a selective calcium-signal-stimulating system (similar to a transistor in an electronic circuit) that can potentially trigger calcium influx and thereby activate calcium-sensitive promoters such as c-fos, BDNF, or NFAT and consequently selectively express target proteins for use in various biomedical applications such as cardiac and/or neuroscience research. It is confirmed that the EPG is related to the control of Ca ion channels but the exact pathway is unknown. The proper design of gene sequence could be the key to responsiveness to EMP. Further studies regarding possible mechanisms should be performed.