Jennifer A. Tate

Tumor cell targeting by iron oxide nanoparticles is dominated by different factors in vitro versus in vivo.

Realizing the full potential of iron oxide nanoparticles (IONP) for cancer diagnosis and therapy requires selective tumor cell accumulation. Here, we report a systematic analysis of two key determinants for IONP homing to human breast cancers: (i) particle size and (ii) active vs passive targeting. In vitro, molecular targeting to the HER2 receptor was the dominant factor driving cancer cell association. In contrast, size was found to be the key determinant of tumor accumulation in vivo, where molecular targeting increased tumor tissue concentrations for 30 nm but not 100 nm IONP. Similar to the in vitro results, PEGylation did not influence in vivo IONP biodistribution. Thus, the results reported here indicate that the in vitro advantages of molecular targeting may not consistently extend to pre-clinical in vivo settings. These observations may have important implications for the design and clinical translation of advanced, multifunctional, IONP platforms.

MAGNETIC NANOPARTICLE HYPERTHERMIA IN CANCER TREATMENT

The activation of magnetic nanoparticles (mNPs) by an alternating magnetic field (AMF) is currently being explored as technique for targeted therapeutic heating of tumors. Various types of superparamagnetic and ferromagnetic particles, with different coatings and targeting agents, allow for tumor site and type specificity. Magnetic nanoparticle hyperthermia is also being studied as an adjuvant to conventional chemotherapy and radiation therapy. This review provides an introduction to some of the relevant biology and materials science involved in the technical development and current and future use of mNP hyperthermia as clinical cancer therapy.

Magnetic nanoparticle hyperthermia: predictive model for temperature distribution

Magnetic nanoparticle (mNP) hyperthermia is a promising adjuvant cancer therapy. mNP’s are delivered intravenously or directly into a tumor, and excited by applying an alternating magnetic field (AMF). The mNP’s are, in many cases, sequestered by cells and packed into endosomes. The proximity of the mNP’s has a strong influence on their ability to heat due to inter-particle magnetic interaction effects. This is an important point to take into account when modeling the mNP’s. Generally, more mNP heating can be achieved using higher magnetic field strengths. The factor which limits the maximum field strength applied to clinically relevant volumes of tissue is the heating caused by eddy currents, which are induced in the noncancerous tissue. A coupled electromagnetic and thermal model has been developed to predict dynamic thermal distributions during AMF treatment. The EM model is based on the method of auxiliary sources and the thermal modeling is based on the Pennes bioheat equation. The results of our phantom study are used to validate the model which takes into account nanoparticle heating, interaction effects, particle spatial distribution, particle size distribution, EM field distribution, and eddy current generation in a controlled environment. Preliminary in vivo data for model validation are also presented. Once fully developed and validated, the model will have applications in experimental design, AMF coil design, and treatment planning.

Imaging and modification of the tumor vascular barrier for improvement in magnetic nanoparticle uptake and hyperthermia treatment efficacy.

The predicted success of nanoparticle based cancer therapy is due in part to the presence of the inherent leakiness of the tumor vascular barrier, the so called enhanced permeability and retention (EPR) effect. Although the EPR effect is present in varying degrees in many tumors, it has not resulted in the consistent level of nanoparticle-tumor uptake enhancement that was initially predicted. Magnetic/iron oxide nanoparticles (mNPs) have many positive qualities, including their inert/nontoxic nature, the ability to be produced in various sizes, the ability to be activated by a deeply penetrating and nontoxic magnetic field resulting in cell-specific cytotoxic heating, and the ability to be successfully coated with a wide variety of functional coatings. However, at this time, the delivery of adequate numbers of nanoparticles to the tumor site via systemic administration remains challenging. Ionizing radiation, cisplatinum chemotherapy, external static magnetic fields and vascular disrupting agents are being used to modify the tumor environment/vasculature barrier to improve mNP uptake in tumors and subsequently tumor treatment. Preliminary studies suggest use of these modalities, individually, can result in mNP uptake improvements in the 3-10 fold range. Ongoing studies show promise of even greater tumor uptake enhancement when these methods are combined. The level and location of mNP/Fe in blood and normal/tumor tissue is assessed via histopathological methods (confocal, light and electron microscopy, histochemical iron staining, fluorescent labeling, TEM) and ICP-MS. In order to accurately plan and assess mNP-based therapies in clinical patients, a noninvasive and quantitative imaging technique for the assessment of mNP uptake and biodistribution will be necessary. To address this issue, we examined the use of computed tomography (CT), magnetic resonance imaging (MRI), and Sweep Imaging With Fourier Transformation (SWIFT), an MRI technique which provides a positive iron contrast enhancement and a reduced signal to noise ratio, for effective observation and quantification of Fe/mNP concentrations in the clinical setting.

Assessment of intratumor non-antibody directed iron oxide nanoparticle hyperthermia cancer therapy and antibody directed IONP uptake in murine and human cells.

Hyperthermia, as an independent modality or in combination with standard cancer treatments such as chemotherapy and radiation, has been established in vitro and in vivo as an effective cancer treatment. However, despite efforts over the past 25 years, such therapies have never been optimized or widelyaccepted clinically. Although methods continue to improve, conventionally-delivered heat (RF, ultrasound, microwave etc) can not be delivered in a tumor selective manner. The development of antibody-targeted, or even nontargeted, biocompatible iron oxide nanoparticles (IONP) now allows delivery of cytotoxic heat to individual cancer cells. Using a murine mouse mammary adenocarcinoma (MTGB) and human colon carcinoma (HT29) cells, we studied the biology and treatment of IONP hyperthermia tumor treatment. Methods: Cancer cells (1 x 106) with or without iron oxide nanoparticles (IONP) were studied in culture or in vivo via implanted subcutaneously in female C3H mice, Tumors were grown to a treatment size of 150 mm3 and tumors volumes were measured using standard 3-D caliper measurement techniques. Mouse tumors were heated via delivery of an alternating magnetic field, which activated the nanoparticles, using a cooled 36 mm diameter square copper tube induction coil which provided optimal heating in 1.5 cm wide region of the coil. The IONPs were dextran coated and had a hydrodynamic radius of approximately 100 nm. For the in vivo studies, intra-tumor, peritumor and rectal (core body) temperatures were continually measured throughout the treatment period. Results: Although some eddy current heating was generated in non-target tissues at the higher field strengths, our preliminary IONP hyperthermia studies show that whole mouse AMF exposure @160 KHz and 400 or 550 Oe, for a 20 minutes (heat-up and protocol heating), provides a safe and efficacious tumor treatment. Initial electron and light microscopic studies (in vitro and in vivo) showed the 100 nm used in our studies are rapidly taken up and retained by the tumor cells. Additional in vitro studies suggest antibodies can significantly enhance the cellular uptake of IONPs.

Intratumoral iron oxide nanoparticle hyperthermia and radiation cancer treatment

The potential synergism and benefit of combined hyperthermia and radiation for cancer treatment is well established, but has yet to be optimized clinically. Specifically, the delivery of heat via external arrays /applicators or interstitial antennas has not demonstrated the spatial precision or specificity necessary to achieve appropriate a highly positive therapeutic ratio. Recently, antibody directed and possibly even non-antibody directed iron oxide nanoparticle hyperthermia has shown significant promise as a tumor treatment modality. Our studies are designed to determine the effects (safety and efficacy) of iron oxide nanoparticle hyperthermia and external beam radiation in a murine breast cancer model. Methods: MTG-B murine breast cancer cells (1 x 106) were implanted subcutaneous in 7 week-old female C3H/HeJ mice and grown to a treatment size of 150 mm3 +/- 50 mm3. Tumors were then injected locally with iron oxide nanoparticles and heated via an alternating magnetic field (AMF) generator operated at approximately 160 kHz and 400 - 550 Oe. Tumor growth was monitored daily using standard 3-D caliper measurement technique and formula. specific Mouse tumors were heated using a cooled, 36 mm diameter square copper tube induction coil which provided optimal heating in a 1 cm wide region in the center of the coil. Double dextran coated 80 nm iron oxide nanoparticles (Triton Biosystems) were used in all studies. Intra-tumor, peri-tumor and rectal (core body) temperatures were continually measured throughout the treatment period. Results: Preliminary in vivo nanoparticle-AMF hyperthermia (167 KHz and 400 or 550 Oe) studies demonstrated dose responsive cytotoxicity which enhanced the effects of external beam radiation. AMF associated eddy currents resulted in nonspecific temperature increases in exposed tissues which did not contain nanoparticles, however these effects were minor and not injurious to the mice. These studies also suggest that iron oxide nanoparticle hyperthermia is more effective than non-nanoparticle tumor heating techniques when similar thermal doses are applied. Initial electron and light microscopy studies of iron oxide nanoparticle and AMF exposed tumor cells show a rapid uptake of particles and acute cytotoxicity following AMF exposure.

Comparison of Iron Oxide Nanoparticle and Waterbath Hyperthermia Cytotoxicity

The development of medical grade iron oxide nanoparticles (IONP) has renewed interest in hyperthermia cancer therapy. Because of their modifiable size and heating capabilities under an AC magnetic field (alternating magnetic field, AMF), IONPs have the potential to damage or kill cells in a manner more therapeutically efficient than previous hyperthermia techniques. The use of IONPs in hyperthermia cancer therapy has prompted numerous questions regarding the cytotoxic mechanism associated with IONP heat therapy and if such mechanism is different (more or less effective) with respect to conventional hyperthermia techniques. In this in vitro study, we determine the immediate and long-term (24 hours) cytotoxic effects of isothermal IONP hyperthermia treatment versus a conventional global heating technique (water bath). Using the same heating time and temperature we showed significantly greater cytotoxicity in IONP-heated cells as opposed to water bath-treated cells. We postulate that the difference in treatment efficacy is due to the spatial relationship of particle-induced thermal damage within cells. Although the exact mechanism is still unclear, it appears likely that intracellular IONPs have to achieve a very high temperature in order to heat the surrounding environment; therefore it is reasonable to assume that particles localized to specific areas of the cell such as the membrane can deliver exacerbated injury to those areas. In this experiment, although detectable global temperature for the particle-heated cells stands comparable to the conventional heat treatment, particle-induced cell death is higher. From the results of this study, we propose that the mechanism of IONP hyperthermia renders enhanced cytotoxicity compared to conventional waterbath hyperthermia at the same measured thermal dose.

Iron oxide nanoparticle enhancement of radiation cytotoxicity

Iron oxide nanoparticles (IONPs) have been investigated as a promising means for inducing tumor cell-specific hyperthermia. Although the ability to generate and use nanoparticles that are biocompatible, tumor specific, and have the ability to produce adequate cytotoxic heat is very promising, significant preclinical and clinical development will be required for clinical efficacy. At this time it appears using IONP-induced hyperthermia as an adjunct to conventional cancer therapeutics, rather than as an independent treatment, will provide the initial IONP clinical treatment. Due to their high-Z characteristics, another option is to use intracellular IONPs to enhance radiation therapy without excitation with AMF (production of heat). To test this concept IONPs were added to cell culture media at a concentration of 0.2 mg Fe/mL and incubated with murine breast adenocarcinoma (MTG-B) cells for either 48 or 72 hours. Extracellular iron was then removed and all cells were irradiated at 4 Gy. Although samples incubated with IONPs for 48 hrs did not demonstrate enhanced post-irradiation cytotoxicity as compared to the non-IONP-containing cells, cells incubated with IONPs for 72 hours, which contained 40% more Fe than 48 hr incubated cells, showed a 25% decrease in clonogenic survival compared to their non-IONP-containing counterparts. These results suggest that a critical concentration of intracellular IONPs is necessary for enhancing radiation cytotoxicity.

Toxicity and Biodistribution of Activated and Non-activated Intravenous Iron Oxide Nanoparticles.

The use of nanoparticles in medical treatment has prompted the question of their safety. In this study, the pathophysiology and biodistribution of three different concentrations of intravenously-delivered dextran-coated Fe3O4 iron oxide nanoparticles (IONP) were evaluated in mice. Some groups of mice were exposed to an AC magnetic field (AMF) at levels comparable with those proposed for cancer treatments. Iron biodistribution analysis for both AMF and non-AMF treated mice was performed for all three concentrations used (.6 mg Fe/mouse, 1.8 mg Fe/mouse, and 5.6 mg Fe/mouse). Blood urea nitrogen, alanine transaminase, alkaline phosphatase, total serum protein, and creatinine were also assessed at 4 hours, 7 days, and 14 days post-injection. Histological analysis of lung, spleen, heart, liver, and kidney tissue was conducted at 7 and 14 days post-injection. Prussian blue and H&E stains were used to histomorphometrically assess iron content in the tissues studied. Preliminary results demonstrate small temporary elevation in liver enzymes and hepatocyte vacuolization at all iron concentrations studied. Liver and spleen were the primary sites of IONP deposition. None of the animals demonstrated systemic or local toxicity or illness, with or without AMF activation.

Development of a biodegradable iron oxide nanoparticle gel for tumor bed therapy.

Treatments of the post-operative surgical bed have proven appealing as the majority of cancer recurrence following tumor resection occurs at the tumor margin. A novel, biodegradable pullulan-based gel infused with magnetic iron oxide nanoparticles (IONP) is presented here for surgical bed administration followed by hyperthermia therapy via alternating magnetic field (AMF) activation. Pullulan is a water soluble, film-forming starch polymer that degrades at the postoperative wound site to deliver the IONP payload, targeting the remaining cancer cells. Different gel formulations containing various % wt of pullulan were tested for IONP elution. Elution levels and amount of gel degradation were measured by immersing the gel in de-ionized water for one hour then measuring particle concentrations in the supernatant and the mass of the remaining gel formulation. The most promising gel formulations will be tested in a murine model of surgical bed resection to assess in vivo gel dissolution, IONP cell uptake kinetics via histology and TEM analysis, and heating capability of the gel with AMF exposure.