Cancer Treatment

How Magnetoelectric Nanoparticles (MENPs) Use Nanoscale Physics to Treat Cancer Cells With Unprecedented High Specificity

To underscore the game changing role of MENPs to treat cancers, it is important to remind of the significant capabilities of electric fields to significantly reduce cancer cell division or entirely eradicate cancer cells with very high specificity, i.e., without affecting the surrounding healthy cells. Unfortunately, to date, these capabilities have been barely exploited because of the challenges to scale down the electric field energy. As you will see shortly, MENPs unlock this enormous potential of the electric fields by leveraging nanotechnology.

It has been established that biological cells respond to electric fields because of the presence of intracellular electrical polarized elements, e.g., cell membranes and  cell-division-partaking proteins such as tubulin and septin. Being electrically polarized,  these elements need to be oriented in a certain way by intrinsic electric fields to maintain certain fundamental functions, such as cell division, within adequate norms. In the cancer cells, these functions do not comply with the norms, and the intrinsic fields, whether at the membrane or in the intracellular space, are not capable of restoring the functions. Consequently, the purpose of any electric-field based technology would be to artificially apply the right electric fields to bring the cellular functions back to normal.

These artificially applied electric fields can be either relatively weak fields to control the cell division or relatively high electric fields to completely eradicate cancer cells, both in a certain frequency range. The strength and frequency of the field would depend on a specific purpose and a cancer type.

There are a number of biological processes which can be controlled by application of an electric field. An example of a high field process is electroporation.  In the electroporation process, they use relatively high electric fields to deliver drugs across the cancer cell membrane, while sparing the healthy cells. An example of a low field process is the reduction of the cancer cell division by aligning the electrically polarized proteins, fundamentally involved in the cell division process, in a certain way by applying relatively low a.c. electric fields in a certain frequency range, e.g., in the hundreds of MHz range.

Electroporation: Application of a sufficiently strong electric field “electroporates” cell membranes (made of electrically polarized lipid bilayers). Specifically, the electric field creates transient localized conductive pores in the membrane. In turn, the formation of transient pores significantly increases permeability of the membrane to biomolecules. This externally controlled transient permeable state of the membrane can be used to deliver vital drugs/biomolecules into the cytosol (intracellular space) for therapeutic purposes. The interpretation of the process, for the first time studied by E. Neumann for gene transfer in 1982, has evolved with more understanding in recent decades. This transition of the membrane’s electric state from non-conducting to conducting happens when the applied electric field exceeds a certain threshold known as the electroporation threshold.

Figure 1. Illustration the lipid bilayer configuration of the cellular membrane with no electric field, E, applied.

Figure 2. Illustration of the formation of a pore in the cellular membrane in response to application of an electric field exceeding the electroporation threshold.

Figs. 1 and 2 illustrate what the cell membranes look like with no field applied and an electric field, E, with a strength exceeding the electroporation threshold, respectively.  Before the field is applied, the membrane is non conducting (Fig. 1) . However, as the field exceeds the threshold value, relatively small pores (~<0.5 nm) are formed through local re-arrangement of lipid molecules in the membrane bilayer so that the lipid heads fold over to form a pore with hydrophilic walls (Fig. 2). In turn, the formation of the conductive water-filled pores effectively increasing the permeability of the membrane. As a result, during the electroporation, biomolecules from the extracellular and intracellular spaces can cross the membrane. Therefore, the electroporation can be used to deliver specific biomolecules to treat cells, e.g., to eradicate cancer.  Furthermore, as described below, because of the molecular level of this underlying mechanism, this externally field-controlled drug delivery can be made highly specific to specific cells only.

The origin of cellular specificity:   The electroporation field threshold scales with the cell membrane potential. The larger the potential is, the larger the threshold is. Consequently, because each cell type has its own distinct membrane potential value, the field-controlled electroporation can be made specific to the cell type and thus used as an enabler of a high specificity treatment. For example, most cancer cells have significantly smaller potentials (and, consequently, electroporation threshold fields) compared to those of their healthy counterparts (Fig. 3 (Figure 1 from the review article by M. Yang and W. Brackenburry, Frontiers in Physiology 4, 185, 2013). Therefore, if the applied field is chosen above the threshold for the cancer cells while  below the threshold for the healthy cells, the cancer cells become electroporated, while the surrounding healthy cells remain intact. Today, the process of electroporation is a recognized approach to provide high specificity drug delivery to treat cancer cells.

Figure 3. Figure 1 from the review article by M. Yang and W. Brackenburry, Frontiers in Physiology 4, 185, 2013.

There are two electroporation types, depending on the electric field strength. They are known as reversible and irreversible electroporation processes, respectively. For example, application of a pulsed electric field on the order of 1000 V/cm with a cycle time on the order of 1 msec can provide adequate conditions to create transient conducting pores which only insignificantly change cellular electrolytes, mostly calcium-based. In this case, turning off the field rapidly eliminates the pores, thus restoring the initial cell state. In other words, the electroporation is a reversible process. However, if the electric field significantly exceeds the threshold value, say by a factor of 10, the electroporation process becomes irreversible. In this case, the electroporated portion of the membrane surface becomes too large, thus leading to significant electrolyte disturbances, in turn resulting in cell death and necrosis. Accordingly, the two electroporation processes can be used for high specificity drug delivery and cell eradication, respectively.

Challenge With Existing Electric Field Driven Therapies:   Despite the aforementioned capabilities, there is a major stumbling block in using electric fields to control biological processes, whether it is by low or high electric fields. It is difficult or maybe even impossible to wirelessly control electric fields, so that the treatment is localized at the cellular level. Remotely initiated electric fields interfere with the electric charges within cells globally and thus cannot be used to control them just locally.  For example, that is why in the existing electroporation approach they use physical electrodes to induce relatively strong electric fields required for the electroporation physics to work.   However, using high-voltage electrodes leads to many limitations including need of surgical intervention, a restricted number of “entry” points, and presence of side effects.   For example, they apply electric fields on the order of 100 V/cm to a relatively large region of the body. As a result, not just cancer cells but also all the healthy cells in the exposed region experience such high electric fields. The energy involved to carry the high fields into the relatively large region is also relatively high and thus can lead to severe side effects. Furthermore, the exposure of healthy cells to such high fields severely limits the efficacy of the electroporation approach. That is why there are other approaches in which they use substantially weaker electric fields to minimize the collateral damage. For example, they use weak a.c. electric fields in a certain frequency range to slow down the division of cancer cells by remotely polarizing tubulin and septin. However, this approach also lacks the localization required for containing the effect to the cancer cells only, thus significantly limiting the therapeutic efficacy.

That is exactly where CNMI’s technology fills in the gap.

Why MENPs are Unique:  As described above, electric fields can be used to provide cell-specific treatment. Depending on a cancer type and a specific approach, e.g., gene transfer, eradication of cancer cells, or cell division rate  reduction,   electric fields of different strengths and frequencies need to be applied.

However, global application of an electric field, regardless of its strength and frequency, makes it difficult to limit the desired electric field effects to cancer cells only, which in turn severely limits the therapeutic efficacy.  In contrast, with MENPs, magnetic fields can be used to control local electric fields in the vicinity of the nanoparticles only. The desired electric field parameters can be generated only in the nanoscale vicinity of the nanoparticles, i.e., in an extremely small volume, compared to the traditional direct applications of electric fields.  As a result, only with a small fraction of the energy (~1/1000), same level electric field effects can be achieved locally, i.e., only in the regions of interest. In turn, this implies the potential to further increase the therapeutic efficacy by orders of magnitude, without causing any side effects.

Unlike any other nanoparticles known to date, MENPs display the so called magnetoelectric (ME) effect at room temperature. Owing to this ME effect, they can provide this “magical” transformation of remotely sourced magnetic fields into local electric fields. This implies that MENPs can combine advantages of the two fields, electric and magnetic, respectively, mitigating each fields’ drawbacks.  For comparison, magnetic fields, unlike electric fields, can freely propagate through the entire body and be localized to a target site without using physical electrodes, though they cannot directly affect the electric properties of the cellular membrane or intracellular proteins responsible for cell proliferation. In contrast, as described above, electric fields can directly affect intrinsic cellular properties, though they cannot propagate through the body without experiencing significant interference with the rest of the “electric circuitry” of the cellular microenvironment. Due to the ME effect, when exposed to magnetic fields, MENPs can generate local electric fields in the locations determined by the magnetic field application. Thus, the desired treatment by either low or high electric fields, can be achieved locally, thus effectively significantly increasing the treatment efficacy.

Figure 4. Physics of MENPs. M and H stand for the magnetization and the magnetic field, respectively. P and E stand for the polarization and the electric field, respectively. The High-resolution TEM image on the right shows a perfect crystal lattice matched interface between the core and the shell. (See Fig. 3 in Nano Lett 20 (8): 5765, 2020.)

The “Magic” of Magnetoelectric Effect Displayed by MENPs:  Again, unlike any other nanoparticles, MENPs display the ME effect. Due to the ME effect, these nanoparticles simultaneously display electric and magnetic effects. Furthermore, these two fields are intrinsically coupled, this coupling is key to the CNMI's technology. In the type of MENPs currently used by CNMI, the coupling is due to the crystal lattice matched interface between the magnetostrictive core and the piezoelectric shell. A high-resolution transmission electron microscopy (TEM) image of a MENP with a net size on the order of 20 nm is shown in Fig. 4. This type of MENPs is known as coreshell MENPs. The relationship between the electric and magnetic fields in these nanostructures is described by the phenomenological expression known as Landau equation:

P = α H,   (1)

where P and H stand for the polarization (electric dipole moment per unit volume) and the magnetic field, respectively.  The linear relationship between the two fields is given by the coefficient α known as the ME coefficient. The expression evaluates the induced  electric dipole moment of the nanoparticle in response to application of a magnetic field.  Reciprocally, application of a local electric field induces a magnetic moment within the nanoparticles, which in turn could be measured via sensitive magnetometers. The reciprocal equation describes the so called converse ME effect:

M = α E,   (2)

Where M and E stand for the magnetization (magnetic dipole moment per unit volume) and the electric field, respectively.  In this case, by convention, the coefficient α is known as the converse ME coefficient. Below, we show how the described physics of MENPs is used to enable high specificity targeted cancer treatments and diagnostics.

Figure 5. Physics of field-controled MENPs-based "Nano-Electroporation" of cancer cells, while sparing the surrounding healthy cells.

MENPs-based Nano-Electroporation for High-Specificity Cancer Cell Targeting:  Due to the above magnetoelectric physics of MENPs, if a magnetic field is applied to the nanoparticles, then they induce their electric field, according to Equation 1. If this electric field, exceeds the electroporation threshold for the selected specific cancer cell type (Fig. 3), then if the nanoparticles are in the nanoscale vicinity of the cell membrane, they will trigger the above electroporation process, now locally. The locality is defined by the size of the nanoparticles. Figure 5 illustrates how MENPs in the vicinity of cell membrane locally trigger electroporation. Because this electroporation is local, i.e., only at the sites close to the nanoparticles, we call this process “Nano-electroporation.” To ensure the MENPs-based targeting has specificity to the specific cancer cells only, the applied magnetic field should not exceed the electroporation threshold for the surrounding healthy cells.  It is noteworthy that after the nanoparticles penetrate the cancer cells only, while sparing the healthy cells, they can either directly eradicate the cancer cells via application of a different type of signals, without using any chemical reagents, or, optionally, release a biological load, e.g., drugs, to activate the eradication of the host (cancer cells).

Figure 6. Confocal microscopy imaging of MDR cells, MES-SA/DX5 line, after they were exposed to the four treatments, (A) control: no drug, (B) control: Flutax-2, (C) control: MENPs loaded with Flutax-2 with no field applied, and (D) MENPs loaded with Flutax-2 with a 30-Oe d.c. magnetic field applied. (See Fig. 4 in Appl Phys Rev 4, 021101.)

Example: Field-controlled Targeting of Multi-drug Resistant (MDR) Cancer Cells:   In the below experiment, using the above physics, MENPs are shown to penetrate multi-drug resistant cancer cells, MES-SA/DX5 line, for their subsequent eradication. It is noteworthy that MDR cells are not easy to eradicate with traditional therapies because of their acquired resistance to drugs. In contrast, with MENPs, the resistance is broken because the nanoparticles, unlike the traditional biological antitumor reagents, use electric field physics to penetrate the membrane and thus can bypass the biological barriers, such as the drug resistant proteins overexpressed by the cancer cells. In this experiment, the MDR cell line was subjected to four different mechanisms to deliver Flutax-2, a fluorescent version of the popular mitotic inhibitor Paclitaxel (aka Taxol) (Fig. 6). The results of confocal microscopy imaging of the MDR cells after they were exposed to the four treatments, (i) control: no drug, (ii) control: Flutax-2, (iii) control: MENPs loaded with Flutax-2 with no field applied, and (iv) MENPs loaded with Flutax-2 with a 30-Oe d.c. magnetic field applied, are shown in Figs. 6a-d, respectively. (The details of the experiment are described in our earlier paper (Stimphil et al, Appl. Phys. Rev. 2017). Quantitatively, the drug uptakes in the four cases were 0, <0.3 %, <0.2 %, and >6 %, respectively. One can clearly see that the drug  uptake in the case of field controlled MENPs was at least an order of magnitude better compared to any of the methods.

Example: Field-controlled Drug Release off MENPs:   One of the fundamental problems of the current targeted drug delivery systems is their relatively poor control of the drug release at the target site. It can be noted that releasing the drug off the carrier nanoparticle, after delivering it to the target site, is critical to restore the bioactivity of the drug. Owing to the ability to control local intrinsic electric fields, MENPs solve this problem. Often, the bond between the drug carrying nanoparticles and their biological loads, e.g., drugs, has an electrostatic or hydrogen nature. It can be noted the both interaction types are electric field based; the electrostatic bond relies on the Coulomb attraction between two opposite charges, the hydrogen bond is due to the attraction of two electric dipoles. Consequently, the both interactions can be controlled by the electric fields of MENPs, which in turn are controlled remotely by a magnetic field. In turn, we use this physics to ensure that the nanoparticle-drug bond is sufficiently strong so that the drug does not fall off in the extracellular space before it reaches the target site.  Only when at the target site, a remote magnetic field command is sent to release the drug. The unique physics of MENPs allow to generate local electric fields of different strengths and frequencies to provide various commands. For example, the delivery process can be achieved via application of d.c. magnetic field gradients, while the controlled release is achieved via application of a special a.c. magnetic field sequence. This physics was described in detail in our earlier publications where MENPs were used to deliver and release special drugs on demand at the target site. For example, the target site can be either a HIV1 virus hidden deep in the brain or metastasized cancer cells.

Figure 7. A simplified illustration of the MENPs-based physics to release a loaded drug off the nanoparticles via application of an a.c. magnetic field. (A) At zero field, the electrostatic nanoparticle-drug (AZTTP) bond is not affected. (B). An additional dipole moment, due to the ME effect, breaks the original symmetry of the charge distribution around the shell. (C) As the field is increased above the bond-specific threshold, the bond on one side is broken. (D,E) The field is reversed to break the bond on the opposite side of the nanoparticle. The red arrows show the electric dipole induced by a magnetic field due to to the ME effect.  (See Fig. 1 in Nat Comm 4: 1707, 2013.)

In Fig. 7, the physics of the a.c. magnetic field controlled release of a drug off MENPs is illustrated using the example of antiretroviral therapy AZTTP loaded on the nanoparticles using a relatively strong electrostatic bond. Due to the ME effect of MENPs, when at the target site, application of an a.c. magnetic field of a 10 Oe with a frequency of 100 Hz effectively “shakes off” the drug of the nanoparticles. In Fig. 8, high resolution atomic force microscopy (AFM) imaging directly shows the magnetic field controlled of the electrostatic bond in action.

Figure 8. High-resolution AFM images which directly trace the key steps of the experiment to demonstrate the physics of an a.c.-magnetic field triggered release of a drug (AZTTP) off a MENP: (A) MENPs and (B) AZTTP molecular chains before the loading (binding) of the drug, (C) AZTTP-MENP nanoformulations after the loading process. Every particle has a molecular chain on its surface. (D) MENPs and (E) AZTTP after the drug release via application of an a.c. magnetic field with a strength of 44 Oe and a frequency of 1000 Hz. (See Fig. 6 in Nat Comm 4: 1707, 2013.)

It is noteworthy that because of the fundamental physics of this intrinsic electric field control, with nanoscale precision, this MENPs-based approach can be extended to any cancer and any drug.  In contrast, the traditional chemotherapy and other approaches are specific to biological microenvironments, thus making them difficult to be extended to any specific cancer. Below, we describe examples in which the above unique electromagnetic physics of MENPs was used to treat various cancers both in in vitro and in vivo models. The underlying physics of the two-step process was described in detail in our previous peer-reviewed paper (Phys. Rev., 4 (2), 021101 (2017)).

Figure 9. Illustration of the two-step process to target ovarian cancer cells with drug-loaded MENPs and then release the drug (Paclitaxel) off the nanoparticles via application of d.c. and a.c. fields, respectively. (a) Initial state: no field is applied (H=0). (b) High-specificity targeting: a d.c. field above the nanoelectroporation (NEP) threshold of the cancer cells is applied, while the field is smaller than the NEP threshold for the healthy cells. (c) Drug release on demand: an a.c. field above the release value is applied to release the drug, thus increasing its bioactivity. (See Fig. 1 in Sci Reports 3: 2953, 2013.)

Treating Ovarian Cancer With MENPs:  The deadliest gynecological cancer, ovarian cancer (OC) is difficult to treat because of the relatively low specificity of the existing treatments. Despite the recently improved survival rates by using intraperitoneal (IP) delivery of anti-neoplastic drugs through surgically implanted catheter, the catheter complications and toxicity have precluded widespread adoption of this invasive means of delivery. Therefore, the importance of using MENPs to provide high-specificity targeted delivery is hard to overestimate.  According to the chart shown in Fig. 2, the ovarian cancer and healthy cells have the membrane potential values of approximately -5 and -50 mV, respectively.  Therefore, by tailoring the properties of MENPs so that they can induce local d.c. electric fields to overcome the membrane potential difference in the range between 5 and 50 mV, drug-loaded MENPs can penetrate the cancer cell membrane, while sparing the surrounding healthy counterparts. Due to the ME effect of MENPs, this tailoring is accomplished by application of a specific d.c. magnetic field. After the drug-loaded nanoparticles end up inside the cancer cells as a result of the nano-electroporation process, an a.c. magnetic field is applied. As explained above, the purpose of the a.c. field is to send a command to release the drug off the nanoparticles and thus activate the drug’s bioactivity to eradicate the host cancer cells. This two-step process of targeted high specificity delivery and release drug via application of d.c. and a.c. magnetic fields, respectively, is described in detail in our papers on treatment of OC in in vitro and in vivo models (Sci Reports 3: 2953, 2013;  Sci Reports 6: 20867, 2016; Phys. Rev., 4 (2), 021101, 2017). The process is illustrated in Fig. 9.

Figure 10. A nude mouse treated with the two-step MENPs process. (a) Tumor photographs at its peak on July 11 (268 mm3) and on October 13 (no visible tumor). (b) IR images (with fluorescent agent Her2Sense 645 taken before (top) and after the completion of the MENPs treatment. The agent had excitation and emission maxima at 643 and 661 nm, respectively. (See Figure 3 in Sci Reports 6: 20867, 2016.)

This two-step MENPs-based physics process was applied to cure nude mice with ovarian cancer cell line SKOV-3 xenografts. The detailed results of the successful treatment study were described in our earlier peer-reviewed paper (Sci Reports 6: 20867, 2016). Fig. 10 shows photographs and near-infrared (NIR) images of a mouse with OC xenografts before and after treatment. The NIR images were taken after administration of a NIR tagged antibody targeting the tumor.  The treatment was conducted through weekly intravenous (IV) administration of a dose of paclitaxel-loaded MENPs, with a subsequent two-step treatment via application of d.c. and a.c. magnetic fields,  for a period of two months. Both primary and metastized cancer cells were eliminated as a result of the treatment. Using energy-dispersive spectroscopy (EDS), an extremely detailed elemental-level compositional analysis of the administrated nanoparticles, showed that nanoparticles indeed targeted the cancer cells with very high specificity (Fig. 11).

Figure 11. SEM-EDS detection of MENPs in the tumor site of a mouse at an initial treatment stage. (left) Regular SEM image of the tumore site, (middle) Barium SEM-EDS and (right) Titanium SEM-EDS scans. Ba and Ti are two key elements representing MENPs. (See Figure 3 in Sci Reports 6: 20867, 2016.)

This experiment proved that MENPs could target cancer cells with relatively high specificity. Similar to the  Enhanced Permeability and Retention (EPR) effect, due to the high leakiness of cancer blood vessels and the lack of a lymphatic system for drainage, the  nanoparticles naturally target the tumor sites. In addition, due to the described fundamental physics, MENPs have the ability for an even higher targeted specificity because of the ability to distinguish between cancer and surrounding healthy cells based on their electric field properties. This experiment also showed that even relatively small metastastized tumors, e.g., in spleen and kidneys, were targeted by MENPs, while sparing all the surrounding healthy cells.

As discussed above, this method is different from traditional approaches, such as chemotherapy and immunotherapy, which are sensitive to intrinsic biological mechanisms. In contrast, because of its fundamental physics-based nature, the MENPs-based targeting can be extended to any cancers and any drugs.

Figure 12. Confocal images showing the specific interaction of MENPs with malignant glioblastoma cells (U87). Cells are stained with DAPI. MENPs are tagged with FITC. FITC-MENPs are specifically associated with U-87MG cells. Uptake is not evident in nonmalignant HBMECs. FITC: Fluorescein isothiocyanate; HBMEC: Human brain microvascular endothelial cell. (See Fig. 6 in Nanomedicine (London) 13 (4): 423, 2018)

Example: In Vitro Study to Deliver Anti-tumor Peptides to Glioblastomas: We and others have shown that nanoparticles, including MENPs, can be used to deliver life-saving drugs across the blood-brain barrier (BBB) (Nanomedicine (London) 10 (13), 2051, 2015; Nat Comm 4: 1707, 2013.). Being sufficiently small (< 50 nm), these nanoparticles easily cross BBB via application of a magnetic field gradient on the order of 1000 V/cm.  Therefore, MENPs represent a powerful tool to deliver vital medicine to treat deadly brain tumors. Externally controlled anticancer effects of binding tumor growth inhibiting synthetic peptides to MENPs were demonstrated in our previous peer-reviewed publication (Nanomedicine (London) 13 (4): 423, 2018)).  MENPs were loaded with molecules of growth hormone releasing hormone antagonist of the MIA class (MIA690), developed by Dr. Andrew Schally. The in vitro experiments used human glioblastoma cells (U-87MG) and human brain microvascular endothelial cells as controls. The studies showed targeted specificity to glioblastoma cells and on-demand release of the peptide by application of the above two-step MENPs’ physics (Fig. 12).

Clearance and Biodistribution of MENPs: A detailed study on the clearance and biodistribution of MENPs in mice was described in our earlier peer-reviewed publication (Nanomedicine (London) 12 (15); 1801, 2017). The study showed that the nanoparticles were naturally excreted within two months after their administration.


Physics Based Cancer Diagnosis

According to the physics of the converse ME effect (Equation 2), if MENPs are exposed to a local electric field, their magnetic moment changes linearly with the electric field. In turn, every cell type has its own signature electric field configuration. The cellular membrane potential is a good example of an intrinsic property which is determined by the specific cell electric field configuration . As illustrated in the chart in Fig. 3, cancer cells have different values of the membrane potential compared to their healthy counterparts.  The reason for the membrane electric field difference between cancer and healthy cells is the difference in their membrane lipid bilayer configurations. In turn, the differently polarized lipids generate different local electric fields in the vicinity of the cancer and healthy cell membranes. These fields can differ by at least an order of magnitude depending on the cancer type and stage. Again, owing to the converse ME effect, this electric field difference can be detected as the magnetic moment change of MENPs. For example, in one previous study, nuclear magnetic resonance (NMR) was used to differentiate the MENPs’ response for different cancer and healthy cells (Scientific Reports 7: 1610, 2017). In general, any magnetometry approach, e.g., vibrating sample magnetometry (VSM), alternating gradient magnetometry (AGM), susceptibility meter, tunneling magnetoresistance (TMR), optical pump magnetometry (OPM), or others could be used to detect the minute electric field change due to the cancer cell type and stage.