Brandon T. Cisneros

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

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.

Remotely Triggered Cisplatin Release from Carbon Nanocapsules by Radiofrequency Fields

The efficacy of nanoparticle-mediated drug delivery is limited by its peri-vascular sequestration, thus necessitating a strategy to trigger drug release from such intra-tumoral nanocarrier-drug depots. In our efforts to explore remotely-activated nanocarriers, we have developed carbon nanocapsules comprised of an ultra-short carbon nanotube shell (US-tubes) loaded with cisplatin (CDDP@US-tubes) and covered with a Pluronic surfactant wrapping to minimize passive release. We demonstrate here that non-invasive radiofrequency (RF) field activation of the CDDP@US-tubes produces heat that causes Pluronic disruption which triggers cisplatin release in an RF-dependent manner. Furthermore, release-dependent cytotoxicity is demonstrated in human hepatocellular carcinoma cell lines.

Cellular uptake and imaging studies of gadolinium‐loaded single‐walled carbon nanotubes as MRI contrast agents

We quantify here, for the first time, the intracellular uptake (J774A.1 murine macrophage cells) of gadolinium-loaded ultra-short single-walled carbon nanotubes (gadonanotubes or GNTs) in a 3 T MRI scanner using R(2) and R(2)* mapping in vitro. GNT-labeled cells exhibited high and linear changes in net transverse relaxations (ΔR(2) and ΔR 2*) with increasing cell concentration. The measured ΔR(2)* were about three to four times greater than the respective ΔR(2) for each cell concentration. The intracellular uptake of GNTs was validated with inductively coupled plasma optical emission spectrometry (ICP-OES), indicating an average cellular uptake of 0.44 ± 0.09 pg Gd per cell or 1.69 × 10(9) Gd(3+) ions per cell. Cell proliferation MTS assays demonstrated that the cells were effectively labeled, without cytotoxicity, for GNTs concentrations ≤28 µM Gd. In vivo relaxometry of a subcutaneously-injected GNT-labeled cell pellet in a mouse was also demonstrated at 3 T. Finally, the pronounced R(2)* effect of GNT-labeled cells enabled successful in vitro visualization of labeled cells at 9.4 T.

Gold nanoparticles and radiofrequency in experimental models for hepatocellular carcinoma

UNLABELLED Hepatocellular carcinoma (HCC) is one of the most lethal and chemo-refractory cancers, clearly, alternative treatment strategies are needed. We utilized 10nm 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. FROM THE CLINICAL EDITOR In a model of hepatocellular carcinoma, the authors demonstrate that non-invasive RF-induced hyperthermia applied with cetuximab targeted delivery of Au NP-gemcitabine conjugate is more effective and safe at dosages ~ 275-fold lower than the current clinically-used systemic dose of gemcitabine.

Stable confinement of positron emission tomography and magnetic resonance agents within carbon nanotubes for bimodal imaging

AIMS Simultaneous positron emission tomography/MRI has recently been introduced to the clinic and dual positron emission tomography/MRI probes are rare and of growing interest. We have developed a strategy for producing multimodal probes based on a carbon nanotube platform without the use of chelating ligands. MATERIALS & METHODS Gd(3+) and (64)Cu(2+) ions were loaded into ultra-short single-walled carbon nanotubes by sonication. Normal, tumor-free athymic nude mice were injected intravenously with the probe and imaged over 48 h. RESULTS & CONCLUSION The probe was stable for up to 24 h when challenged with phosphate-buffered saline and mouse serum. Positron emission tomography imaging also confirmed the stability of the probe in vivo for up to 48 h. The probe was quickly cleared from circulation, with enhanced accumulation in the lungs. Stable encapsulation of contrast agents within ultra-short single-walled carbon nanotubes represents a new strategy for the design of advanced imaging probes with variable multimodal imaging capabilities.

A New Imaging Platform for Visualizing Biological Effects of Non-Invasive Radiofrequency Electric-Field Cancer Hyperthermia.

Herein, we present a novel imaging platform to study the biological effects of non-invasive radiofrequency (RF) electric field cancer hyperthermia. This system allows for real-time in vivo intravital microscopy (IVM) imaging of radiofrequency-induced biological alterations such as changes in vessel structure and drug perfusion. Our results indicate that the IVM system is able to handle exposure to high-power electric-fields without inducing significant hardware damage or imaging artifacts. Furthermore, short durations of low-power (< 200 W) radiofrequency exposure increased transport and perfusion of fluorescent tracers into the tumors at temperatures below 41°C. Vessel deformations and blood coagulation were seen for tumor temperatures around 44°C. These results highlight the use of our integrated IVM-RF imaging platform as a powerful new tool to visualize the dynamics and interplay between radiofrequency energy and biological tissues, organs, and tumors.

Hyperthermia Inhibits Recombination Repair of Gemcitabine-Stalled Replication Forks

BACKGROUND Gemcitabine is a potent nucleoside analogue against solid tumors, but development of drug resistance is a substantial problem. Removal of gemcitabine incorporated into DNA by repair mechanisms may contribute to resistance in chemo-refractory solid tumors. Human hepatocellular carcinoma (HCC) is usually very chemoresistant to gemcitabine. METHODS We treated HCC in vitro and in vivo (orthotopic murine model with human Hep3B or HepG2 xenografts, 7-10 CB17SCID mice per group) with gemcitabine. The role of homologous recombination repair proteins in repairing stalled replication forks was evaluated with hyperthermia exposure and cell-cycle analysis. The Student t-test was used for two-sample comparisons. Multiple group data were analyzed using one-way analysis of variance. All statistical tests were two-sided. RESULTS We demonstrated that Mre11-mediated homologous recombination repair of gemcitabine-stalled replication forks is crucial to survival of HCC cells. Furthermore, we demonstrated inhibition of Mre11 by an exonuclease inhibitor or concomitant hyperthermia. In orthotopic murine models of chemoresistant HCC, the Hep3B tumor mass with radiofrequency plus gemcitabine treatment (mean ± SD, 180±91mg) was statistically significantly smaller compared with gemcitabine alone (661±419mg, P = .0063). CONCLUSIONS This study provides mechanistic understanding of homologous recombination inhibiting-strategies, such as noninvasive radiofrequency field-induced hyperthermia, to overcome resistance to gemcitabine in refractory human solid tumors.

Radiofrequency electric-field heating behaviors of highly enriched semiconducting and metallic single-walled carbon nanotubes

It is theorized that enhanced thermal heating may result from exposing single-walled carbon nanotubes (SWNTs) embedded in a conductive host to radiofrequency (RF) electric fields. We examine the RF-induced (13.56 MHz) heating behaviors of 95% metallic- and semiconducting-enriched SWNTs (m-/s-SWNTs) suspended in aqueous solutions with varying NaCl molarity (0.001 mM–1 M). The heating effects were only evident for host molarities below 1 mM (equivalent to 0.1 S/m) at which the s-SWNT heating rates dominated those of the m-SWNTs. The heating effects were localized to aligned and aggregated “SWNT ropes” ~1 cm in length that formed in suspension, parallel to the electric-field vector, during the RF exposure. For molarities above 1 mM, no enhancements were evident, owing to the large heating effects of the bulk ionic NaCl suspensions, which were observed in previous studies. Although larger enhancement effects proportional to the host conductivity have been theoretically predicted for m-/s-SWNT suspensions, this was not observed most likely because of the aggregation and screening effects, which diminished the scattered electric field near the m-/s-SWNTs. Our research may further the development of better nanoparticle heating agents for applications such as non-invasive RF-induced cancer hyperthermia.

Cytotoxicity and variant cellular internalization behavior of water-soluble sulfonated nanographene sheets in liver cancer cells

Highly exfoliated sulfonated graphene sheets (SGSs), an alternative to graphene oxide and graphene derivatives, were synthesized, characterized, and applied to liver cancer cells in vitro. Cytotoxicity profiles were obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, WST-1[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, and lactate dehydrogenase release colorimetric assays. These particles were found to be non-toxic across the concentration range of 0.1 to 10 μg/ml. Internalization of SGSs was also studied by means of optical and electron microscopy. Although not conclusive, high-resolution transmission and scanning electron microscopy revealed variant internalization behaviors where some of the SGS became folded and compartmentalized into tight bundles within cellular organelles. The ability for liver cancer cells to internalize, fold, and compartmentalize graphene structures is a phenomenon not previously documented for graphene cell biology and should be further investigated.

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis. In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.