Noninvasive Radiofrequency Field Research Articles

Radiofrequency-Activated Pyroptosis of Bi-Valent Gold Nanocluster for Cancer Immunotherapy.

Pyroptosis is gasdermin-mediated programmed necrosis that exhibits promising potential application in cancer immunotherapy, and the main challenge lies in how to provoke specific pyroptosis of tumor cells. Here, biGC@PNA with a precisely stoichiometric ratio of Au(I) ion/Au(0) atom induced pyroptosis of tumor cells by its radiofrequency (RF)-heating effect. An in vitro/in vivo assay on 4T1 tumor cells indicates RF-activated pyroptosis of tumor cells elicits a robust ICD effect, enhancing the synergistic antitumor efficacy of biGC@PNA with decitabine, significantly suppressing tumor metastasis and relapse by provoking systemic antitumor immune responses. Utilizing RF-activated pyroptotic immune responses, biGC@PNA efficiently enhances the antitumor efficacy of αPD-1 immunotherapy under RF irradiation and provides a promising strategy for improving cancer immunotherapy by the noninvasive RF field with high clinical transformation potential.

Ultrarapid Inductive Rewarming of Vitrified Biomaterials with Thin Metal Forms

Arteries with 1-mm thick walls can be successfully vitrified by loading cryoprotective agents (CPAs) such as VS55 (8.4 M) or less concentrated DP6 (6 M) and cooling at or beyond their critical cooling rates of 2.5 and 40 °C/min, respectively. Successful warming from this vitrified state, however, can be challenging. For example, convective warming by simple warm-bath immersion achieves 70 °C/min, which is faster than VS55’s critical warming rate of 55 °C/min, but remains far below that of DP6 (185 °C/min). Here we present a new method that can dramatically increase the warming rates within either a solution or tissue by inductively warming commercially available metal components placed within solutions or in proximity to tissues with non-invasive radiofrequency fields (360 kHz, 20 kA/m). Directly measured warming rates within solutions exceeded 1000 °C/min with specific absorption rates (W/g) of 100, 450 and 1000 for copper foam, aluminum foil, and nitinol mesh, respectively. As proof of principle, a carotid artery diffusively loaded with VS55 and DP6 CPA was successfully warmed with high viability using aluminum foil, while standard convection failed for the DP6 loaded tissue. Modeling suggests this approach can improve warming in tissues up to 4-mm thick where diffusive loading of CPA may be incomplete. Finally, this technology is not dependent on the size of the system and should therefore scale up where convection cannot.

Non-Invasive Radiofrequency Field Treatment of 4T1 Breast Tumors Induces T-cell Dependent Inflammatory Response

Previous work using non-invasive radiofrequency field treatment (RFT) in cancer has demonstrated its therapeutic potential as it can increase intratumoral blood perfusion, localization of intravenously delivered drugs, and promote a hyperthermic intratumoral state. Despite the well-known immunologic benefits that febrile hyperthermia can induce, an investigation of how RFT could modulate the intra-tumoral immune microenvironment had not been studied. Thus, using an established 4T1 breast cancer model in immune competent mice, we demonstrate that RFT induces a transient, localized, and T-cell dependent intratumoral inflammatory response. More specifically we show that multi- and singlet-dose RFT promote an increase in tumor volume in immune competent Balb/c mice, which does not occur in athymic nude models. Further leukocyte subset analysis at 24, 48, and 120 hours after a single RFT show a rapid increase in tumoral trafficking of CD4+ and CD8+ T-cells 24 hours post-treatment. Additional serum cytokine analysis reveals an increase in numerous pro-inflammatory cytokines and chemokines associated with enhanced T-cell trafficking. Overall, these data demonstrate that non-invasive RFT could be an effective immunomodulatory strategy in solid tumors, especially for enhancing the tumoral trafficking of lymphocytes, which is currently a major hindrance of numerous cancer immunotherapeutic strategies.

Non-Invasive Radiofrequency Field Treatment to Produce Hepatic Hyperthermia: Efficacy and Safety in Swine.

The Kanzius non-invasive radio-frequency hyperthermia system (KNiRFH) has been investigated as a treatment option for hepatic hyperthermia cancer therapy. The treatment involves exposing the patient to an external high-power RF (13.56 MHz) electric field, whereby the propagating waves penetrate deep into the tumor causing targeted heating based on differential tissue dielectric properties. However, a comprehensive examination of the Kanzius system alongside any associated toxicities and its ability to induce hepatic hyperthermia in larger animal models, such as swine, are the subjects of the work herein. Ten Yucatan female mini-swine were treated with the KNiRFH system. Two of the pigs were treated a total of 17 times over a five-week period to evaluate short- and long-term KNiRFH-associated toxicities. The remaining eight pigs were subjected to single exposure sessions to evaluate heating efficacy in liver tissue. Our goal was to achieve a liver target temperature of 43°C and to evaluate toxicities and burns post-treatment. Potential toxicities were evaluated by contrast-enhanced MRI of the upper abdomen and blood work, including complete metabolic panel, complete blood count, and liver enzymes. The permittivities of subcutaneous fat and liver were also measured, which were used to calculate tissue specific absorption rates (SAR). Our results indicate negligible KNiRFH-associated toxicities; however, due to fat overheating, liver tissue temperature did not exceed 38.5°C. This experimental limitation was corroborated by tissue permittivity and SAR calculations of subcutaneous fat and liver. Significant steps must be taken to either reduce subcutaneous fat heating or increase localized heating, potentially through the use of KNiRFH-active nanomaterials, such as gold nanoparticles or single-walled carbon nanotubes, which have previously shown promising results in murine cancer models.

Intravital microscopy for evaluating tumor perfusion of nanoparticles exposed to non-invasive radiofrequency electric fields.

Poor biodistribution and accumulation of chemotherapeutics in tumors due to limitations on diffusive transport and high intra-tumoral pressures (Jain RK, Nat Med. 7(9):987–989, 2001) have prompted the investigation of adjunctive therapies to improve treatment outcomes. Hyperthermia has been widely applied in attempts to meet this need, but it is limited in its ability to reach tumors in deeply located body regions. High-intensity radiofrequency (RF) electric fields have the potential to overcome such barriers enhancing delivery and extravasation of chemotherapeutics. However, due to factors, including tumor heterogeneity and lack of kinetic information, there is insufficient understanding of time-resolved interaction between RF fields and tumor vasculature, drug molecules and nanoparticle (NP) vectors. Intravital microscopy (IVM) provides time-resolved high-definition images of specific tumor microenvironments, overcoming heterogeneity issues, and can be integrated with a portable RF device to enable detailed observation over time of the effects of the RF field on kinetics and biodistribution at the microvascular level. Herein, we provide a protocol describing the safe integration of IVM with a high-powered non-invasive RF field applied to 4T1 orthotopic breast tumors in live mice. Results show increased perfusion of NPs in microvasculature upon RF hyperthermia treatment and increased perfusion, release and spreading of injected reagents preferentially in irregular vessels during RF exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12645-016-0016-7) contains supplementary material, which is available to authorized users.

Gold-gold sulfide nanoshell as a novel intensifier for anti-tumor effects of radiofrequency fields.

Several studies have been carried out to investigate the effect of various nanoparticles exposed to radiofrequency (RF) waves on cancerous tissues. In this study, a colon carcinoma tumor model was irradiated by RF in the presence of gold-gold sulfide (GGS) nanoshells. Synthesis and characterization of GGS nanoshells were initially performed. CT26 cells were subcutaneously injected into the flank of BALB/c mice to create the colon carcinoma tumor models. Then the tumors were subjected to different treatments. Treatment factors included intratumoral injection of GGS and RF radiation. Different groups were considered as control with no treatment, receiving GGS, RF irradiated and simultaneous administration of GGS and RF. Efficacy of the treatments was evaluated by daily monitoring of tumor volume and recording the relative changes in it, the time needed for a 5-fold increase in the volume of tumor (T5) and utilizing pathologic studies to determine the lost volume of the tumors. In comparison with control group, tumor growth was not markedly inhibited in the groups receiving only GGS or RF, while in the group receiving GGS and RF, tumor growth was effectively inhibited compared with the other groups. In addition, the lost volume of the tumor and T5 was markedly higher in groups receiving GGS and RF compared with other groups. This study showed that RF radiation can markedly reduce the tumor growth in presence of GGS. Hence, it can be predicted that GGS nanoshells convert sub-lethal effects of noninvasive RF fields into lethal damages.

Autophagy and enhanced chemosensitivity in experimental pancreatic cancers induced by noninvasive radiofrequency field treatment

Patients with pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options and poor response to the standard gemcitabine (GCB)-based chemotherapy. In the current study, the authors investigated the feasibility of noninvasive short-wave radiofrequency (RF) electric fields to improve the cytotoxic effect of GCB on PDAC cells and determined its mechanism of action. The cytotoxicity of RF alone and in combination with GCB was studied in vitro on normal pancreatic human pancreatic ductal epithelial cells and different PDAC cell lines by flow cytometry, and in vivo on ectopic and orthotopic human PDAC xenograft models in mice. The mechanism of RF activity was studied by Western blot analysis and immunohistochemistry. Toxicity was determined by histopathology. Exposure of different PDAC cells to 13.56-megahertz radio waves resulted in a substantial cytotoxic effect, which was accompanied by the induction of autophagy but not apoptosis. These effects of RF were found to be absent in normal cells. Excessive numbers of autophagosomes in cancer cells persisted 24 to 48 hours after RF exposure and then declined. The addition of a subtoxic dose of GCB to RF treatment inhibited the recovery of cancer cells from the RF-induced autophagy and enhanced the cytotoxic effect of the latter on cancer cells. The treatment of PDAC in situ in mice with the combination of noninvasive RF and GCB was found to have a superior antitumor effect compared with the use of RF or GCB alone, yet there was no evidence of systemic toxicity. Noninvasive RF treatment induced autophagy but not apoptosis in cancer cells and demonstrated potential as an enhancer of chemotherapy for treating patients with pancreatic cancer without toxicity to normal cells.

Noninvasive Radiofrequency Field Destruction of Pancreatic Adenocarcinoma Xenografts Treated with Targeted Gold Nanoparticles

Pancreatic carcinoma is one of the deadliest cancers with few effective treatments. Gold nanoparticles (AuNP) are potentially therapeutic because of the safety demonstrated thus far and their physiochemical characteristics. We used the astounding heating rates of AuNPs in nonionizing radiofrequency (RF) radiation to investigate human pancreatic xenograft destruction in a murine model. Weekly, Panc-1 and Capan-1 human pancreatic carcinoma xenografts in immunocompromised mice were exposed to an RF field 36 hours after treatment (intraperitoneal) with cetuximab- or PAM4 antibody-conjugated AuNPs, respectively. Tumor sizes were measured weekly, whereas necrosis and cleaved caspase-3 were investigated with hematoxylin-eosin staining and immunofluorescence, respectively. In addition, AuNP internalization and cytotoxicity were investigated in vitro with confocal microscopy and flow cytometry, respectively. Panc-1 cells demonstrated increased apoptosis with decreased viability after treatment with cetuximab-conjugated AuNPs and RF field exposure (P = 0.00005). Differences in xenograft volumes were observed within 2 weeks of initiating therapy. Cetuximab- and PAM4-conjugated AuNPs demonstrated RF field-induced destruction of Panc-1 and Capan-1 pancreatic carcinoma xenografts after 6 weeks of weekly treatment (P = 0.004 and P = 0.035, respectively). There was no evidence of injury to murine organs. Cleaved caspase-3 and necrosis were both increased in treated tumors. This study demonstrates a potentially novel cancer therapy by noninvasively inducing intracellular hyperthermia with targeted AuNPs in an RF field. While the therapy is dependent on the specificity of the targeting antibody, normal tissues were without toxicity despite systemic therapy and whole-body RF field exposure.

Pancreatic carcinoma cells are susceptible to noninvasive radio frequency fields after treatment with targeted gold nanoparticles

Gold and carbon nanoparticles absorb nonionizing radio frequency (RF) energy and release heat. Solid gold nanoparticles are delivered to cancer cells via conjugation with targeting antibodies. Here, 20-nm gold particles were conjugated to cetuximab, which is an epidermal growth factor receptor-1 (EGFR-1) antibody. A pancreatic carcinoma cell line that highly expresses EGFR-1, Panc-1, and Cama-1, which is a breast carcinoma cell line that minimally expresses EGFR-1, were treated with 100-nmol/L cetuximab-conjugated gold nanoparticles for 3 h (n = 4). Thirty-six hours later, the dishes were placed in an RF field with a generator power of 200 W for 5 min. After another 36 h, cell injury and death were evaluated with flow cytometry. The targeted cell line Panc-1 had a viability of 46% +/- 12%, whereas the Cama-1 cell had a viability of 92% +/- 2% after RF field exposure (P < .008). Transmission electron microscopy showed gold nanoparticle uptake in Panc-1 cells but negligible uptake by Cama-1 cells. Nontargeted cells do not internalize a sufficient amount of antibody-conjugated gold nanoparticles to induce injury in a noninvasive RF field. This technique could be useful in cancer treatment if a cancer-specific antibody is used to localize gold nanoparticles to malignant cells.

Abstract 3704: Gemtuzumab ozogamicin conjugated gold nanoparticles induce cell death in D1.1 leukemia cells when exposed to a radiofrequency field

Abstract Introduction: Acute T-cell leukemia cells may express the marker CD33. Gemtuzumab is a monoclonal antibody against CD33, used clinically in combination with ozogamicin, a cytotoxic antibiotic, for treatment of acute leukemias. It has been previously demonstrated that gold nanoparticles delivered intracellularly and exposed to nonionizing radiofrequency (RF) fields induce hyperthermic cytotoxicity. RF fields are not cytotoxic without the presence of nanoparticles. The purpose of this experiment is to investigate cell death in cells treated with gemtuzumab ozogamicin conjugated to gold nanoparticles after RF field exposure. Methods: D1.1 (acute T-cell leukemia) cells were plated in 60mm cell culture dishes. Two groups (n=3 in each group) of plated cells were treated for two hours with gemtuzumab ozogamicin conjugated to 10nm solid gold nanoparticles in media. One group of cells was then exposed to a radiofrequency field for a total generator energy of 1500 W-minutes. The control cells were treated with the same gold conjugate, but were not exposed to an RF field. Two days later, cell viability, apoptosis, and death was determined by flow cytometry. The groups were compared with Student's t-test. Results: RF fields induced apoptosis in 58.7% ± 3.95% of treated cells, compared with 8.66% ± 0.56% of the controls (p=0.002). The viability after RF exposure was 13.9% ± 6.11% while gemtuzumab ozogamicin gold conjugate alone decreased viability to 74.2% ± 0.85% (p=0.003). The temperature of the media was kept below 40°C to avoid nonspecific cell injury. Conclusions: Multimodality therapy (chemotherapy and nanoparticle-mediated hyperthermia) induced significantly more death in acute T-cell leukemia cells than antibody-chemotherapy alone. Antibodies specific to molecular targets on cells permit targeting of the heat mediator (nanoparticles) while preventing non-specific delivery of both nanoparticles and chemotherapeutic agent. The use of non-invasive RF fields to specifically induce cell death by hyperthermia with nanoparticles may present new options for the treatment of leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3704.

Noninvasive Radiofrequency Field Therapy Induces Cell Death in Human Pancreatic Carcinoma Cells Targeted with Gold Nanoparticles

Noninvasive Radiofrequency Field Therapy Induces Cell Death in Human Pancreatic Carcinoma Cells Targeted with Gold Nanoparticles

Abstract C99: Panitumumab conjugated gold nanoparticles induce hyperthermic cytotoxicity in pancreatic carcinoma cell lines after noninvasive radiofrequency field exposure

Abstract Introduction: Many cancer cells that highly express epidermal growth factor receptor-1 (EGFR-1) have been demonstrated to endocytose antibody specific targeted nanoparticles such as cetuximab conjugated gold nanoparticles. These gold nanoparticles are subsequently delivered throughout the cell. When gold nanoparticles are exposed to near infrared laser (NIR) radiation or non-invasive radiofrequency fields, they heat in a concentration dependent manner. Radiofrequency (RF) fields are advantageous because they easily penetrate through body tissues without injury. We investigated the effects of a clinically available EGFR-1 antibody, panitumumab, conjugated to gold nanoparticles in noninvasive RF fields. Methods: Solid gold nanoparticles (10 nm) were covalently linked to panitumumab, a monoclonal antibody against EGFR-1. Two pancreatic carcinoma cell lines, Panc-1 and Capan-1, were treated with 200 nM panitumumab linked gold nanoparticles in media for four hours. After replacing the media with fresh media not containing the antibody linked gold nanoparticles, the cells were exposed to an RF field at a generator power of 600W for a total of 3 minutes. The media temperature was kept below 42°C in order to prevent non-specific apoptosis. Viability and apoptosis were evaluated with flow cytometry three days later. Statistical analysis was performed with the Student's t-test and uncertainties represent standard errors of the mean. Results: The viability of control cells not treated with the conjugate remained greater than 90% for both cell lines. The antibody-nanoparticle conjugate alone decreased the viability of Panc-1 cells to 81.0% ± 1.1%. However, after RF field exposure, the viability decreased to 57.2% ± 7.9% (p &amp;lt; 0.03). After antibody-conjugate treatment, Capan-1 cells viability was 89.5% ± 3.9%. After RF field exposure, however, the viability decreased to 67.8% ± 3.8% (p &amp;lt;0.03). Conclusions: The susceptibility of pancreatic carcinoma cell lines to gold nanoparticle mediated noninvasive RF field hyperthermic therapy may be related to the combined effects of monoclonal antibody treatment and intracellular hyperthermia. Based on this simple model, it appears that there is a relationship between EGFR-1 expression, cytotoxicity associated with monoclonal antibody therapy, and hyperthermic cytotoxicity after RF field exposure. Combined rational therapy utilizing non-invasive RF fields may provide for opportunities where traditional radiotherapy has failed and NIR therapy is not readily possible due to tumor location deep within a body cavity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C99.

Carbon nanotube‐enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field

Single-walled carbon nanotubes (SWNTs) have remarkable physicochemical properties that may have several medical applications. The authors have discovered a novel property of SWNTs-heat release in a radiofrequency (RF) field-that they hypothesized may be used to produce thermal cytotoxicity in malignant cells. Functionalized, water-soluble SWNTs were exposed to a noninvasive, 13.56-megahertz RF field, and heating characteristics were measured with infrared thermography. Three human cancer cell lines were incubated with various concentrations of SWNTs and then treated in the RF field. Cytotoxicity was measured by fluorescence-activated cell sorting. Hepatic VX2 tumors in rabbits were injected with SWNTs or with control solutions and were treated in the RF field. Tumors were harvested 48 hours later to assess viability. The RF field induced efficient heating of aqueous suspensions of SWNTs. This phenomenon was used to produce a noninvasive, selective, and SWNT concentration-dependent thermal destruction in vitro of human cancer cells that contained internalized SWNTs. Direct intratumoral injection of SWNTs in vivo followed by immediate RF field treatment was tolerated well by rabbits bearing hepatic VX2 tumors. At 48 hours, all SWNT-treated tumors demonstrated complete necrosis, whereas control tumors that were treated with RF without SWNTs remained completely viable. Tumors that were injected with SWNTs but were not treated with RF also were viable. The current results suggested that SWNTs targeted to cancer cells may allow noninvasive RF field treatments to produce lethal thermal injury to the malignant cells. Now, the authors are developing SWNTs coupled with cancer cell-targeting agents to enhance SWNT uptake by cancer cells while limiting uptake by normal cells.