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

Summary

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

Abstract

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.

Introduction

The absorption of RF energy by biological tissue (due to their inherent electrical permittivity) results in elevated tissue temperatures as a function of time, which eventually leads to cell death by hyperthermia. It is hypothesized that cancer hyperthermia can be optimized through the use of targeted nanomaterials that internalize within the cancer cell and act as RF-thermal transducers, leaving the neighboring healthy, normal cells intact. Several reports have already shown that a variety of NPs can act as effective RF heat sources which aid in cancer necrosis^1-4.

In these regards, gold NPs (AuNPs)^3-5, carbo....

Protocol

A complete experimental overview is depicted in Figure 1.

Further details are depicted in steps 1-3 below.

1. Assessing RF Heating of NP Colloids: AuNPs as an Example

1. In general, for each NP sample being investigated, first wash the sample several times through a centrifugation filter with deionized (DI) water to remove background ions and contaminants. All ions and contaminants will have been removed from the AuNP suspension when the liquid being washed out has similar RF heating rates (HRs) as DI water. This purification process also allows for higher concentrations of NPs to be obt....

Results

1. Assessing RF heating of NP colloids: AuNPs as an example.

After following section 1.1 - 1.2.3 expect to have a highly concentrated, stable, and purified solution of 5 nm and 10 nm diameter AuNPs. From the 500 ml as-purchased stock solution, expect to obtain at least 4 ml of solution at a concentration of 1,000 mg/L. The difference in HRs between the AuNPs and the background DI water buffer solution at this concentration should be ~0.25 °C/sec and 0.1 °C/sec for 5 nm and 10 nm AuNPs, respective.......

Discussion

These protocols allow the experimentalist to fully analyze the extent to which nanomaterials (in this case AuNPs) can increase RF-induced hyperthermia for cancer treatment. The first protocol specifically deals with analyzing heat production from highly-concentrated and purified AuNP samples. Although other groups have reported heat production primarily from the buffers which the AuNPs are suspended in and not the AuNPs themselves^9-11, their RF systems used lower concentrations of AuNPs with diameters >10 n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
500 ml gold nanoparticles (5 nm)	Ted Pella, INC	15702-5
Amicon Ultra-4/-15 Centrifugal Filter Units (50 kDa)	Millipore	UFC805024/UFC910096	(4 ml and 15 ml volumes)
MEM X1 Cell Culture Media	Cellgro	10-101-CV	(add extra nutrients as necessary)
Fetal Bovine Serum	Sigma	F4135-500 ml
Copper Tape	Ted Pella	16072
Equipment
Kanzius RF System (13.56 MHZ)	ThermMed, LLC, Inc. (Erie, PA, USA)
IR Camera	FLIR SC 6000, FLIR Systems, Inc. (Boston, MA, USA)	Contact FLIR
1.3 ml Quartz Cuvette	ThermMed, LLC, Inc. (Erie, PA, USA)
Teflon Sample holder with Rotary Stage	ThermMed, LLC, Inc. (Erie, PA, USA)
SPECTROstar Nano Microplate reader	BGM Labtech
UV-Vis spectrometer	Applied Nanofluorescence, Houston, TX)	NS1 NanoSpectralyzer
ICP-–S	PerkinElmer	Optima 4300 DV
Zetasizer	Malvern	Zen 3600 Zetasizer