P. J. Hoopes

Magnetic nanoparticle biodistribution following intratumoral administration

Recently, heat generated by iron oxide nanoparticles (IONPs) stimulated by an alternating magnetic field (AMF) has shown promise in the treatment of cancer. To determine the mechanism of nanoparticle-induced cytotoxicity, the physical association of the cancer cells and the nanoparticles must be determined. We have used transmission electron microscopy (TEM) to define the time dependent cellular uptake of intratumorally administered dextran-coated, core-shell configuration IONP having a mean hydrodynamic diameter of 100-130 nm in a murine breast adenocarcinoma cell line (MTG-B) in vivo. Tumors averaging volumes of 115 mm3 were injected with iron oxide nanoparticles. The tumors were then excised and fixed for TEM at time 0.1-120 h post-IONP injection. Intracellular uptake of IONPs was 5.0, 48.8 and 91.1% uptake at one, 2 and 4 h post-injection of IONPs, respectively. This information is essential for the effective use of IONP hyperthermia in cancer treatment.

Fe∕Fe oxide nanocomposite particles with large specific absorption rate for hyperthermia

Using a water-in-oil microemulsion with cetyl trimethyl ammonium bromide as the surfactant, iron was reduced to form a metallic core on which a passivating oxide shell was grown. Transmission electron microscopy, vibrating sample magnetometry, and heating measurements were used to characterize these monodispersed magnetic Fe∕Fe3O4 composite nanoparticles with respect to the possible application for magnetic hyperthermia treatments of cancer. The aim is to utilize the fact that an iron core (high saturation magnetization) will give a greater heating effect than iron oxide, while the iron oxide coating will allow the nanoparticles to be observed using magnetic resonance imaging so that therapy can be effectively monitored and targeted. The largest specific absorption rate obtained was 345W∕g under an alternating magnetic field of 150Oe at 250kHz.

Iron oxide nanoparticle hyperthermia and radiation cancer treatment

It is established that heat can enhance the effect of radiation cancer treatment. Due to the ability to localize thermal energy using nanoparticle hyperthermia, as opposed to other, less targeted, hyperthermia modalities, it appears such enhancement could be accomplished without complications normally associated with systemic or regional hyperthermia. This study employs non-curative (suboptimal), doses of heat and radiation, in an effort to determine the therapeutic enhancement potential for IONP hyperthermia and radiation. Methods: MTG-B murine breast adenocarcinoma cell are inoculated into the right flanks of female CH3/HEJ mice and grown to volumes of 150mm3+ /- 40 mm3. A single dose of 15 Gy (6 MeV) radiation was uniformly delivered to the tumor. A pre-defined thermal dose is delivered by direct injection of iron oxide nanoparticles into the tumor. By adjusting the field strength of the 160 KHz alternating magnetic field (AMF) an intra-tumoral temperature between 41.5 and 43 degrees Celsius was maintained for 10min. The alternating magnetic field was delivered by a water-cooled 36mm diameter square copper tube induction coil operating at 160 kHz with variable magnet field strengths up to 450 Oe . The primary endpoint of the study is the number of days required for the tumor to achieve a volume 3 fold greater than the volume at the time of treatment (tumor regrowth delay). Results: Preliminary results suggest the addition of a modest IONP hyperthermia to 15 Gy radiation achieved an approximate 50% increase in tumor regrowth delay as compared to a 15 Gy radiation treatment alone. The therapeutic effects of IONP heat and radiation combined were considered additive, however in mice that demonstrated complete response (no tumor present after 30 days), the effect was considered superadditive or synergistic. Although this data is very encouraging from a multimodality cancer therapy standpoint, additional temporal and dose related information is clearly necessary to optimize the therapy.

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.

Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

BackgroundUSP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease.MethodsHeterozygous USP18 +/− FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined.ResultsUSP18 −/− FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6–12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441).ConclusionsUSP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy.

Synthesis and heating effect of iron/iron oxide composite and iron oxide nanoparticles

Fe/Fe oxide nanoparticles, in which the core consists of metallic Fe and the shell is composed of Fe oxides, were obtained by reduction of an aqueous solution of FeCl3 within a NaBH4 solution, or, using a water-in-oil micro-emulsion with CTAB as the surfactant. The reduction was performed either in an inert atmosphere or in air, and passivation with air was performed to produce the Fe/Fe3O4 core/shell composite. Phase identification and particle size were determined by X-ray diffraction and TEM. Thermal analysis was performed using a differential scanning calorimeter. The quasistatic magnetic properties were measured using a VSM, and the specific absorption rates (SARs) of both Fe oxide and Fe/Fe3O4 composite nanoparticles either dispersed in methanol or in an epoxy resin were measured by Luxtron fiber temperature sensors in an alternating magnetic field of 150 Oe at 250 kHz. It was found that the preparation conditions, including the concentrations of solutions, the mixing procedure and the heat treatment, influence the particle size, the crystal structure and consequently the magnetic properties of the particles. Compared with Fe oxides, the saturation magnetization (MS) of Fe/Fe3O4 particles (100-190 emu/g) can be twice as high, and the coercivity (HC) can be tunable from several Oe to several hundred Oe. Hence, the SAR of Fe/Fe3O4 composite nanoparticles can be much higher than that of Fe oxides, with a maximum SAR of 345 W/g. The heating behavior is related to the magnetic behavior of the nanoparticles.

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 microwave and magnetic nanoparticle hyperthermia radiosensitization in murine breast tumors

Hyperthermia has been shown to be an effective radiosensitizer. Its utility as a clinical modality has been limited by a minimally selective tumor sensitivity and the inability to be delivered in a tumor-specific manner. Recent in vivo studies (rodent and human) have shown that cancer cell-specific cytotoxicity can be effectively and safely delivered via iron oxide magnetic nanoparticles (mNP) and an appropriately matched noninvasive alternating magnetic field (AMF). To explore the tumor radiosensitization potential of mNP hyperthermia we used a syngeneic mouse breast cancer model, dextran-coated 110 nm hydrodynamic diameter mNP and a 169 kHz / 450 Oe (35.8 kA/m) AMF. Intradermally implanted (flank) tumors (150 ± 40 mm3) were treated by injection of 0.04 ml mNP (7.5 mg Fe) / cm3 into the tumor and an AMF (35.8 kA/m and 169 kHz) exposure necessary to achieve a CEM (cumulative equivalent minute) thermal dose of 60 (CEM 60). Tumors were treated with mNP hyperthermia (CEM 60), radiation alone (15 Gy, single dose) and in combination. Compared to the radiation and heat alone treatments, the combined treatment resulted in a greater than two-fold increase in tumor regrowth tripling time (tumor treatment efficacy). None of the treatments resulted in significant normal tissue toxicity or morbidity. Studies were also conducted to compare the radiosensitization effect of mNP hyperthermia with that of microwave-induced hyperthermia. The effects of incubation of nanoparticles within tumors (to allow nanoparticles to be endocytosed) before application of AMF and radiation were determined. This preliminary information suggests cancer cell specific hyperthermia (i.e. antibody-directed or anatomically-directed mNP) is capable of providing significantly greater radiosensitization / therapeutic ratio enhancement than other forms of hyperthermia delivery.

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 hyperthermia and chemotherapy cancer treatment

The benefit of combining hyperthermia and chemotherapy to treat cancer is well established. However, combined therapy has not yet achieved standard of care status. The reasons are numerous and varied, however the lack of significantly greater tumor cell sensitivity to heat (as compared to normal cells) and the inability to deliver heat to the tumor in a precise manner have been major factors. Iron oxide nanoparticle (IONP) hyperthermia, alone and combined with other modalities, offers a new direction in hyperthermia cancer therapy via improved tumor targeting and an improved therapeutic ratio. Our preliminary studies have demonstrated tumor cell cytotoxicity (in vitro and in vivo) with IONP heat and cisplatinum (CDDP) doses lower than those necessary when using conventional heating techniques or cisplatinum alone. Ongoing studies suggest such treatment could be further improved through the use of targeted nanoparticles.