The goal of this exploratory research is to develop a technique for remote stimulation of mammalian cells using nanoscale electric fields. This new concept takes advantage of magnetoelectric properties of multiferroic nanoparticles which can generate local electric fields in the proximity of cell membranes when subjected to external magnetic field pulses. These fields are expected to control functions of voltage-gated ion channels, which are voltage-sensitive macromolecules, responsible for transport of Na+, K+ ions across cell membranes. To elucidate feasibility of this new approach PI will use computer simulations and thin film technology at the Advanced Materials Research Institute of the University of New Orleans to design and fabricate nanoelectrodes and patterned arrays of multiferroic particles to generate nanoscale electric fields. The response of the ion channels to the nano-electrostimulation will be measured by Co-PI in his Biophysics Laboratory at Loyola University New Orleans using modified patch-clamp technique. Since future in-vivo applications of the new method will involve use of multiferroic nanocomposites of magnetic and ferroelectric nanoparticles, The Co-PI will also devise methods of delivery of the ferroelectric nanoparticles and will determine their toxicity and binding to mammalian cells. This collaborative research will efficiently use resources and expertise in physics, materials science and biophysics available at the University of New Orleans and Loyola University New Orleans.
Intellectual Merit: The proposed research is an attempt to utilize magnetoelectric properties of multiferroic nanoparticles as wireless probes to electrostimulate mammalian cells. The effects of external nanoscale electric fields on ion transport in mammalian cells have not been studied and this research will provide better understanding of fundamental functions of the cells. Although there have been extensive studies on applications of magnetic nanoparticles in biological systems, little is known about the interactions of ferroelectric nanoparticles with mammalian cells and proposed research will shed light on feasibility of biomedical applications of ferroelectric materials and their composites. New methods will be developed for intracellular and extracellular delivery of the nanoparticles, and patch-clamp technique will be modified to test responses of the ion-channels in living cells to applied magnetic fields with the frequency up to 5 kHz. The sequences of the pulses, as well as properties of the nanoparticles will be tuned to detetermine control of ion currents.
Broader Impacts: The outcome of this research on is expected to have profound effect on several disciplines, such as biology, medicine, biotechnology and bionics. Successful control of ion transport using magnetic field pulses offers alternative noninvasive method to treat ion-channel related diseases, such as cystic fibrosis, diabetes, cardiac arrhythmias, neurologic and psychiatric diseases, gastrointestinal disorders, cardiovascular diseases and hypertension. Since voltage-gated ion channels are responsible for triggering and propagation of action potentials in neurons the new mechanism of stimulation of ion channels can be used to treat pain and psychiatric diseases. Ultimately, electric fields from magnetoelectric nanoparticles can be used to interface neurons with bionic devices which can remotely control their action potentials. This project will greatly benefit undergraduate and graduate students participating in this research through extensive training in modern technologies, hands on experiment experience and communication skills gained through participation in meetings and conferences that will impact their future professional careers in academia or industry.
A new application of nanoparticles in biotechnology has been recently proposed [1]. This new mechanism of remote control of ion channels is illustrated in Fig.1. Multiferroic nanoparticles with magnetic core and ferroelectric shell are capable of generating local electric fields when exposed to magnetic field pulse. When placed near the cells, these fields are likely to interact with voltage-gated ion channels, which are macromolecules in cell membranes responsible for ion transport across the cell membranes. This new approach may potentially lead to treatment for many human and animal diseases such as diabetes, cystic fibrosis, cardiac arrhythmias, neurological disorders or hypertension, which result from malfunctioning of ion channels. This grant addressed two issues which had to be resolved prior to attempting to implement the nanoparticles in medical applications. The first was the issue of potential toxicity of ferroelectric nanoparticles, which are components of the multiferroic nanoparticles. Secondly, because little is known about effects of micro- and nano-scale electric fields on physiological functions of cells we investigated the effect of electric fields of microscopic electrodes on ionic currents through ion channels in mammalian cells. The experiments were designed to mimic the action of electric field due to multiferroic nanoparticles. Toxicity tests were conducted on tsa201 cells stably expressing the Shaker K+ channel. The choice of these cells with voltage-gated channels was motivated by their use in electrophysiological experiments in the latter parts of this project. Tests were conducted to determine the effect of two kinds of ferroelectric particles: microparticles of barium titanate (IV) and nanoparticles of barium strontium titanium oxide, on the cell properties and growth rate. Growth media were prepared with varying concentrations of sonicated BT(IV) and BaSTO in suspension. The cells were observed and photographed at t=0, 6, 12, 18, 24, 30, & 36 hours after BaSTO/BT(IV) suspension was added to healthy, established cells. There was no statistically significant effect of exposure to micro and nanoparticle suspensions of different concentrations on growth rates of tsA201 cell lines in vitro (see Fig. 2). It was also observed that the ferroelectric nanoparticles did not tend to aggregate at the cell membranes. Hence, to ensure that the nanoparticles are in close proximity of membrane-bound ion channels, a functionalization of the particles will be required to bind them to the cells, as described in [2]. The response of ion channels in cell membranes to local external electric field was studied using the apparatus shown schematically in Fig. 3. It was based on a patch-clamping technique in which one of the electrodes makes a direct electrical connection with the interior of the cell through a micropipette forming a gigaseal with the membrane, whereas the other electrode is placed in the bath containing extracellular medium in which the cell is immersed. These patch clamp electrodes were used to apply voltage from the Axopatch 200B patch-clamping amplifier to vary cross-membrane polarization and to measure the ionic currents at the same time. The external electric fields were generated using micro-patterned electrodes in close vicinity of the samples. Triangular gold electrodes (see Fig. 4) with the rounded tip of diameter of a few micrometers, separated by a distance of 5 μm (less than the typical cell size), were prepared using magnetron sputtering and optical photolithography. Voltage pulses between electrodes, with no reference to ground potential, mimicked action of the floating potentials due to multiferroic nanoparticles. They were delivered using the battery powered Digimeter DS2A Constant Voltage Isolated Stimulator, marked as IS in Fig. 3. Alternative way of stimulating the external electrodes was to apply signals from a Function Generator (instead of IS) while disconnecting the patch-clamping pipette electrode with a relay and measuring the ion current shortly after reconnecting the relay. A comparison of ionic current measurements with and without external stimulation is presented in Fig. 5. It is evident that the external stimulus from the IS applied to coverslip electrodes causes modulation of the ionic current similar to what can be achieved by simply modifying the control voltage in a voltage-clamp experiment. However, it's worth emphasizing that the modification of ionic currents was in this case achieved using external variation of floating potential with no reference to the bath or cell interior potential. Summarizing, both experiments demonstrated that future progress in remote stimulation of ion channels is possible: no adverse effects of ferroelectric particles on cells in terms of toxicity were observed and possibility of external stimulation of voltage-gated channels by floating potentials was confirmed. Reference [1] A. Kargol, L. Malkinski and G. Caruntu "Biomedical Applications of Multiferroic Nanoparticles" in "Advanced Magnetic Materials" ISBN 978-953-51-0637-1, DOI: 10.5772/2298, ed. L. Malkinski, Intech 2012 ch.4, pp. 89-118. [2] B. Kozissnik and J. Dobson, Biomedical Applications of Mesoscale Magnetic Particles, MRS Bulletin (2013) vol. 38, no. 11 pp. 927-931
