High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature dosages in the tumor area

Abstract

Purpose: Tumor cells can be effectively inactivated by heating mediated by magnetic nanoparticles. However, optimized nanomaterials to supply thermal stress inside the tumor remain to be identified. The present study investigates the therapeutic effects of magnetic hyperthermia induced by superparamagnetic iron oxide nanoparticles on breast (MDA-MB-231) and pancreatic cancer (BxPC-3) xenografts in mice in vivo.

Methods: Superparamagnetic iron oxide nanoparticles, synthesized either via an aqueous (MF66; average core size 12 nm) or an organic route (OD15; average core size 15 nm) are analyzed in terms of their specific absorption rate (SAR), cell uptake and their effectivity in in vivo hyperthermia treatment.

Results: Exceptionally high SAR values ranging from 658 ± 53 W*gFe (-1) for OD15 up to 900 ± 22 W*gFe (-1) for MF66 were determined in an alternating magnetic field (AMF, H = 15.4 kA*m(-1) (19 mT), f = 435 kHz). Conversion of SAR values into system-independent intrinsic loss power (ILP, 6.4 ± 0.5 nH*m(2)*kg(-1) (OD15) and 8.7 ± 0.2 nH*m(2)*kg(-1) (MF66)) confirmed the markedly high heating potential compared to recently published data. Magnetic hyperthermia after intratumoral nanoparticle injection results in dramatically reduced tumor volume in both cancer models, although the applied temperature dosages measured as CEM43T90 (cumulative equivalent minutes at 43°C) are only between 1 and 24 min. Histological analysis of magnetic hyperthermia treated tumor tissue exhibit alterations in cell viability (apoptosis and necrosis) and show a decreased cell proliferation.

Conclusions: Concluding, the studied magnetic nanoparticles lead to extensive cell death in human tumor xenografts and are considered suitable platforms for future hyperthermic studies.

Fig. 1

Experimental workflow of in vivo magnetic hyperthermia experiments. One and seven days after magnetic nanoparticle (MNP) application, magnetic hyperthermia was conducted on subcutaneous xenografts (AMF: H = 15.5 kA*m⁻¹, f = 435 kHz) for 60 min. Tumor volume (V_tumor) and body weight (wt_body) were monitored at the indicated days. Blood count of the animals was determined on day 0, 14 and 28 post first magnetic hyperthermia. AMF alternating magnetic field.

Fig. 2

MNP characterization. (a) TEM micrographs and size distribution. Data was fitted to a lognormal distribution (red line). (b) MNP characteristics including average core size, coating, hydrodynamic diameter (z-average), Polydispersity index (PDI) and zeta potential of OD15 and MF 66. MNP magnetic nanoparticles.

Fig. 3

High SAR values for OD15 and MF66 MNP dispersed in water, agarose and polyvinylalcohol. The two latest reproduce immobilization conditions in vivo. (a) SAR values of both used MNP depending on the viscosity of the media (water, 1% (w/v) agar in water, 10% PVA in DMSO: water (80: 20% (v/v))). Intrinsic loss power (ILP) of OD15 and MF66 dispersed in water, agarose, and PVA. (b) Prussian Blue staining of MDA-MB-231 cells after incubation with 125 pg Fe/cell of OD15 and MF66 for 24 h at 37°C. Bars: 50 μm. (c) Hematoxylin stained tumor tissue of MF66 treated MDA-MB-231 xenografts 3 days after intratumoral application. Nuclei stained blue, MNP stained brown. Bar: 20 μm. Error bars indicate standard deviations. MNP magnetic nanoparticles, PVA polyvinylalcohol hydrogel, SAR specific absorption rate.

Fig. 4

Repeated treatment of MNP bearing tumors in the AMF (H = 15.4 kA*m⁻¹, f = 435 kHz) led to significant reduction of tumor volume compared to untreated ones. Effect of magnetic hyperthermia on the relative tumor volume in the course of a 4 week period after therapy in comparison to untreated tumors for MDA-MB-231 (a) and BxPC-3 xenografts (b). Additionally T90 temperatures and macroscopic tumor images are displayed. (* p ≤ 0.05, ** p ≤ 0.01 (Mann–Whitney-U-Test: treated vs. untreated)). Error bars indicate standard deviations. AMF alternating magnetic field, MNP magnetic nanoparticles, T90 temperature exceeded by 90% of the tumor surface.

Fig. 5

Despite the prevailing of tumor surface temperatures below 43°C, high temperature spots were also present. Temperature distribution maps (heat maps) of tumor surface temperatures of the two consecutive AMF treatments. The percentages of tumor area treated with temperatures below 43°C, between 43 and 45°C and above 45°C plotted against the individual relative tumor volume at 28 days after the first therapy are shown. MDA-MB-231 xenografts treated with magnetic hyperthermia using MF66 (a) or OD15 (b). BxPC-3 xenografts treated with magnetic hyperthermia using MF66 (c) or OD15 (d). AMF alternating magnetic field.

Fig. 6

Neither the treatment within the alternating magnetic field (AMF, H = 15.4 kA*m⁻¹, f = 435 kHz), nor with the magnetic nanoparticles (MNP, ≤0.5 mg Fe per 100 mm³) alone had a significant effect on tumor growth compared to the untreated animals. Relative tumor volume on day 28 of MDA-MB-231 (a) and BxPC-3 xenografts (b) for animals after only AMF or MNP treatment compared to untreated animals. Additionally, relative tumor volumes of magnetic hyperthermia treated animals at 28 days after first magnetic hyperthermia therapy for both xenograft models are displayed. (* p ≤ 0.05, ** p ≤ 0.01 (Mann–Whitney-U-Test: treated vs. untreated)). Error bars indicate standard deviations.

Fig. 7

Magnetic hyperthermia leads to apoptosis and necrosis in tumor tissue. Hematoxylin/eosin stained tumor tissue of magnetic hyperthermia (MF66) treated and untreated MDA-MB-231 xenografts 2 days after first magnetic hyperthermia. (a) Necrotic and apoptotic tissue, (b) Vital tumor tissue, (c) Transition from necrotic and apoptotic to vital tumor tissue indicated by dashed line, (d) Vital untreated tumor tissue. Tissue sections were stained with hematoxylin and eosin. Nuclei stained blue, MNP stained brown. Bars: 50 μm. MNP magnetic nanoparticles.

Fig. 8

Magnetic hyperthermia diminished proliferative activity of tumor tissue compared to untreated tissue. (a) KI-67 staining of untreated and magnetic hyperthermia (MF66) treated MDA-MB-231 tumor tissue extracted 2 days and 28 days after the first magnetic hyperthermia. KI-67 positive cells stained red, nuclei stained blue. Bars: 200 μm. (b) Percentage of KI-67 positive tissue area 2 days and 28 days after the respective treatment. Bars represent categorical distribution of KI-67 positive area within the whole tissue section for all evaluated slides per group. AMF alternating magnetic field, MNP magnetic nanoparticles.

Fig. 9

OD15 and MF66 magnetic hyperthermia treatment did not alter the blood composition, indicating a good biotolerability. The number of white blood cells (*10³*μl⁻¹), red blood cells (*10⁶*μl⁻¹), and the amount of hemoglobin (g*dl⁻¹) are displayed for the magnetic hyperthermia treatment and the three control groups at 0 day, 14 days and 28 days after first hyperthermia. Black dashed lines refer to reference values (Harlan Laboratories, Venray, The Netherlands; http://www.harlan.com). MDA-MB-231 xenografts treated with MF66 (a) or OD15 (b). BxPC-3 xenografts treated with MF66 (c) or OD15 (d). Error bars indicate standard deviations. AMF alternating magnetic field, MNP magnetic nanoparticles.