Gold nanoparticles and radiofrequency in experimental models for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal and chemo-refractory cancers, clearly, alternative treatment strategies are needed. We utilized 10 nm gold nanoparticles as a scaffold to synthesize nanoconjugates bearing a targeting antibody (cetuximab, C225) and gemcitabine. Loading efficiency of gemcitabine on the gold nanoconjugates was 30%. Targeted gold nanoconjugates in combination with RF were selectively cytotoxic to EGFR expressing Hep3B and SNU449 cells when compared to isotype particles with/without RF (P < 0.05). In animal experiments, targeted gold nanoconjugates halted the growth of subcutaneous Hep3B xenografts in combination with RF exposure (P < 0.05). These xenografts also demonstrated increased apoptosis, necrosis and decreased proliferation compared to controls. Normal tissues were unharmed. We have demonstrated that non-invasive RF-induced hyperthermia when combined with targeted delivery of gemcitabine is more effective and safe at dosages ~ 275-fold lower than the current clinically-delivered systemic dose of gemcitabine

Bioorthogonal Tetrazine-Mediated Transfer Reactions Facilitate Reaction Turnover in Nucleic Acid-Templated Detection of MicroRNA

Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates

Tumor Selective Hyperthermia Induced by Short-Wave Capacitively-Coupled RF Electric-Fields

<div><p>There is a renewed interest in developing high-intensity short wave capacitively-coupled radiofrequency (RF) electric-fields for nanoparticle-mediated tumor-targeted hyperthermia. However, the direct thermal effects of such high-intensity electric-fields (13.56 MHZ, 600 W) on normal and tumor tissues are not completely understood. In this study, we investigate the heating behavior and dielectric properties of normal mouse tissues and orthotopically-implanted human hepatocellular and pancreatic carcinoma xenografts. We note tumor-selective hyperthermia (relative to normal mouse tissues) in implanted xenografts that can be explained on the basis of differential dielectric properties. Furthermore, we demonstrate that repeated RF exposure of tumor-bearing mice can result in significant anti-tumor effects compared to control groups without detectable harm to normal mouse tissues.</p></div

E-field measurements.

<p>Electric field measurements were performed using the setup described in methods. The measurements were performed in air or in the peritoneal cavity of mice while keeping the E-field probe at the same <i>x,y,z</i> location, which approximately corresponded with the location of orthotopic tumors in tumored mice. <i>(n = 3, data points represent mean, and error bars represent standard deviation).</i></p

Thermal dose quantification in Hep3B and MDA PATC-3 xenografts under RF field exposure (13.56 MHz, 600 W).

<p><i>Panel A.</i> Hep3B xenograft and normal mouse liver temperatures were measured in real-time using fiber optic thermography while abdominal surface/skin temperatures were measured using infrared thermography. RF exposure was started at a tumor temperature of 35°C. <i>(n = 6). Panel B.</i> MDA PATC-3 xenograft and intra-peritoneal temperatures were measured in real-time using fiber optic thermography while abdominal surface/skin temperatures were measured using infrared thermography. RF exposure was started at a tumor temperature of 37°C. <i>(n = 9). (Solid line represents mean, dashed line represents standard deviation, and vertical dotted line represents frequency of 13.56 MHz).</i></p

Radiofrequency generator and fiber optic probe placement.

<p><i>Panel A.</i> For fiberoptic thermography, a temperature-sensing probe is placed through a 20 G needle. The needle is advanced into the tumor (T) under ultrasound guidance. The probe is then advanced through the needle and the needle is withdrawn. <i>Panel B.</i> Kanzius 13.56 MHz external RF generator system is shown (black box) that is connected to an end-firing antenna in the transmission head (Tx). A spacing of 3.5 inches exists between the Tx head and the receiver head (Rx)/ground plate. <i>Panel C.</i> A CB17 SCID mouse is placed supine on the ground plate of the Rx head. A copper shield made from copper tape is used to ground all mice and prevent electrothermal injury. An abdominal window is created in the middle of the copper shield to allow RF field exposure to the tumor-bearing area.</p

Dielectric properties of tumor tissues.

<p><i>Ex-vivo</i> dielectric spectroscopy of tumors (Hep3B and MDA PATC-3 xenografts) and normal tissues (liver and pancreas) was performed. Permittivity (ε’) and imaginary permittivity (ε”) are shown for each tissue. <i>(n = 4–15, Solid line represents mean, dashed line represents standard deviation, and vertical dotted line represents frequency of 13.56 MHz). To compare permittivity values of normal and tumor tissues, two-tailed unpaired Student’s t-test was performed. p<0.01 for all datasets.</i></p