The MNP used in this study, denominated MF66, were produced by means of the co-precipitation technique . Coating with dimercaptosuccinic acid (DMSA) was performed as described previously [27, 28]. Briefly, the MNP were initially coated with oleic acid and dispersed in toluene and a solution of DMSA in dimethyl sulfoxide (DMSO) was added to perform a ligand exchange from oleic acid to DMSA. The DMSA-coated MNP precipitated and were washed several times with water. Finally, the MNP were resuspended in distilled water, the pH adjusted and sterile filtration carried out. The MF66 MNP used in this study are already characterized and their magnetic properties have been studied . The hydrodynamic diameter was measured by dynamic light scattering (DLS) and expressed as the Z-average size of the MNP dispersed in water. Furthermore, we measured the ζ- potential (Zetasizer Nano ZS, Malvern Instruments) of the MNP at pH 7.4.
For the electrostatic immobilization, either 5.1 μl of an N6L solution (1.93 mM) or 202 μl of a DOX hydrochloride solution (500 μM, Cell pharm, Bad Vilbel, Germany) were incubated with 1 ml MF66 for 6 h at room temperature. N6L multivalent pseudopeptide was synthesized, purified and analyzed for its biological effect as previously described . In the case of N6L, the mixture was purified by ultrafiltration or centrifuged. MF66-DOX was centrifuged and the supernatant was removed. MNP pellets were re-dispersed at 2.4 mg Fe/ml.
To immobilize both DOX and N6L onto MF66 MNP, the same protocol as described above was used; here, DOX was immobilized first onto MF66 MNP followed by N6L. To quantify the amount of immobilized N6L, an N6L fluorescently labeled with Alexa Fluor 546 (N6L-AF546) was synthesized and used for immobilization onto MF66 under the same conditions as described for N6L. The unbound N6L-AF546 recovered during the washes was measured (λexc = 555 nm, λem = 560–750 nm). Similarly, immobilized DOX was quantified by measuring the fluorescence of unbound DOX in the supernatant (λexc = 495 nm, λem = 520–750 nm).
For the preparation of the MNPs used for the in vitro and in vivo studies, the bare MNPs were sonicated for 3 minutes and then filtered through a 0.22-μm strainer for sterilization. The iron concentration was measured after filtration by Inductively coupled plasma – mass spectrometry (ICP-MS) before functionalization. All functionalization processes were then carried out under sterile conditions and all molecules used (N6L or DOX solutions) were filtered through a 0.22-μm strainer.
Specific absorption rate of MNP
To assess the heating potential of the MNP in an AMF (conditions H = 15.4 kA/m, f = 435 kHz) we determined the specific absorption rate (SAR) and intrinsic loss power (ILP) using colorimetric methods as described before .
Release of DOX and N6L-AF546
The mode of the release of the electrostatically immobilized molecules onto MNP was monitored . Water was used to determine the stability of the three formulations over time. Then, the same experiments were performed in PBS buffer (pH 7.4) and in phenol red-free DMEM with 10% (v/v) fetal bovine serum (FBS) (complete DMEM) to modulate desorption of N6L-AF546 and DOX from the MNP in the presence of salts. MNP were dispersed at a final concentration of 0.3 mg Fe/ml. The samples were then placed at 37°C and at different time points (up to 120 h), 100 μl of each sample were collected, centrifuged and supernatants were analyzed by fluorescence and compared to a reference sample.
Three genotypically diverse breast-derived cell lines were used for in vitro testing of the MNP. Two cell lines (MCF-7 and MDA-MB-231, both ATCC) were selected due to their distinct cancer phenotypes. In comparison to the two cancerous cell line models, a third non-cancerous cell line MCF-10A (ATCC, mammary epithelial cells) was used as a control. Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2 and maintained in DMEM with 10% (v/v) FBS and 1% PenStrep (all products from Gibco®, Paisley, Scotland, UK). Cells were tested regularly using the MycoAlert® PLUS test kit (Lonza, Switzerland) for the presence of mycoplasma and prior to freezing stock. All experiments were conducted using sub-confluent cells in the exponential phase of growth. Depending on the experiment, cells were seeded in 24-well or 96-well plates and incubated for 24 h prior to MNP exposure.
MNP internalization and subcellular localization
MDA-MB-231 cells grown on coverslips were incubated for 24 h with MF66, MF66-N6L, MF66-DOX or MF66-N6LDOX (all at 100 μg Fe/ml) in cell culture medium. To remove non-internalized MNP, samples were washed and observed immediately or after 48 h, under bright light for internalization, or by fluorescence microscopy for subcellular location of DOX (n = 3 independent experiments).
Prussian blue staining for iron detection
The presence and localization of iron particles in MDA-MB-231 cells were assessed by Prussian blue staining. Cells were incubated with MNP for 24 h and then analyzed immediately or 48 h post incubation. Cells were washed, fixed in methanol, and stained with equal volumes of 4% hydrochloric acid and 4% potassium ferrocyanide trihydrate (all Panreac Química) for 15 minutes, and counterstained with neutral red.
Impact of nanoparticles on cells in the absence of hyperthermic conditions
Cells were allowed to attach to the culture plate for 24 h and then exposed for 24 h and 72 h to the MNP formulations. Concentrations in the range of 5 to 200 μg Fe/mL were employed to determine if the selected MNP formulation elicited a cytotoxic response in each cell line. Triplicate experiments were conducted with three wells per concentration. Positive and negative controls were included as previously described . Following 24 h incubation, cells were washed, stained for 30 minutes using LysoTracker® (Molecular Probes, Eugene, OR, USA), an indicator for cell membrane permeability, fixed using 3.7% paraformaldehyde (PFA) for 20 minutes and further stained for 10 minutes with Hoechst 33342 nuclear dye (Thermo Fisher Scientific Inc., Waltham, MA, USA). Screening was carried out by high content analysis using the GE Healthcare InCell1000 Analyzer, Buckinghamshire, UK by bright field and three fluorescent channels as described before .
In vitro cell viability determination under hyperthermic conditions
The sensitivity of MDA-MB-231 cells to hyperthermic temperatures and the effect of the MNP formulations on cell viability in the presence and absence of heat were assessed. At 24 h after seeding 5000 cells/well in a 96-well plate (Greiner BioOne, Frickenhausen, Germany), cells were incubated with either medium, MF66, MF66-N6L, MF66-DOX or MF66-N6LDOX in a concentration of 100 μg Fe/ml or the equivalent molar amount of free N6L (400 nM), DOX (4 μM) or N6L and DOX for 24 h at 37°C.
For hyperthermia treatment, cells were put in the incubator at 46°C for 30 minutes, corresponding to a temperature dosage of 90 cumulative equivalent minutes at 43°C (CEM43T90, for details see Additional file 1), or else cells were left in the incubator at 37°C. At 48 h after hyperthermia, cells were washed twice with Hank's balanced salt solution (HBSS), once with medium and then AlamarBlue® reagent was added for 4 h . Fluorescence was measured (Tecan Infinite M1000 Pro, Grödig, Austria; λexc = 530–560 nm, λem = 590 nm) and normalized to the fluorescence of untreated cells (no MNP incubation and no hyperthermia).
Hyperthermia treatment in tumor-bearing athymic nude mice
All in vivo hyperthermia experiments, the experimental workflow, determination of temperature dosage and heat distribution and data analysis were carried out as described before in detail . All experiments were in accordance with international guidelines on the ethical use of animals and were approved by the regional animal care committee (02-068/11, Thüringer Landesamt für Verbraucherschutz, Bad Langensalza, Germany). Animals were maintained under artificial day-night cycles (12 h light-dark cycles; 23°C room temperature, 30%–60% environment humidity) and received food and water ad libitum.
Briefly, to induce xenografts we injected 120 μl Matrigel™ containing 2 × 106 MDA-MB-231 cells subcutaneously on the rear backside of the nude mice and allowed tumor growth until a volume between 100 and 250 mm3 was reached. MNP formulations MF66, MF66-N6L, MF66-DOX or MF66-N6LDOX were used in concentrations of 0.25 mg Fe/100 mm3 for intratumoral injection 24 h prior to the first in vivo hyperthermia treatment (Figure 1). Depending on the tumor size this equaled a concentration range of 0.15–0.375 mg N6L/kg body weight and 0.22–0.55 mg DOX/kg body weight. Hyperthermia treatment were conducted on days 0 and 7. Tumor volume was measured with a caliper every 3 days and compared to the untreated control animals (ddH2O injection, no AMF treatment). The tumors of anesthetized animals were placed inside the coil of the AMF (H = 15.4 kA/m, f = 435 kHz) for magnetic hyperthermia treatment. The tumor surface and rectal temperatures were monitored by fiber optic temperature sensors. To ensure animal safety, we ensured that the temperatures in non-tumor tissue and the rectum did not exceed 38°C during hyperthermia treatment. We calculated the temperature dosage on the tumor surface, namely T90 temperatures and CEM43T90 values according to Sapareto et al.  based on the infrared thermography data. To determine complete tumor regression, a relative tumor volume of 20% was chosen as the cutoff value to account for remaining skin and cicatrical tissue.
Micro computed tomography (µCT) imaging of intratumoral MNP distribution
For optimization of the applied heat distribution on tumors, we analyzed the individual intratumoral MNP distribution during in vivo hyperthermia treatment. Therefore, we conducted µCT imaging of the animals directly after MNP application and for follow up on days 7 and 28 (TomoScope Synergy Twin, CT Imaging, Erlangen, Germany) using a low radiation-dose protocol (29 s, 65 kV). MNP distribution and volume were analyzed with the Imalytics Research Software (Philips Technologie, Aachen, Germany). Using these data, the AMF power was controlled individually to reach 43°C in tumor areas farthest away from MNP deposits.
Iron determination in organs
To investigate MNP biodistribution and degradation, the iron content of tumors and organs was quantified using flame atomic absorption spectroscopy as previously described .
The proliferative behavior of cells depending on magnetic hyperthermia treatment and the MNP formulation was investigated at day 28 after the first magnetic hyperthermia treatment by assessing the Ki67 and Bcl2 protein abundance in paraffin-embedded tissue sections. The degree of vascularization was assessed by CD31 staining. The primary antibodies used were a monoclonal anti-Ki67 antibody (Abcam, Cambridge, UK, 1:500 dilution), a monoclonal mouse anti-human Bcl2 (1:500 dilution, Dako, Hamburg, Germany) and a polyclonal rabbit anti-CD31 (1:500 dilution, Abcam, Cambridge, UK). Antigen detection was visualized via streptavidin-alkaline phosphatase or horseradish peroxidase (for details of staining protocols see Additional file 1). The slides were evaluated by three blinded observers. Ki67/Bcl2-positive areas over the whole tumor sections were evaluated and divided into five categories based on their expression level: (I) no expression to (V) very high expression as described before .
Statistical analysis of cell staining with LysoTracker® was performed as previously described for multiparametric assessment . To determine if differences of tumor volume between animal groups were significant, we conducted the Mann-Whitney U test for analysis of tumor volume and one-way analysis of variance (ANOVA) for in vitro data analysis. For histological differences the Mann-Whitney U test was conducted for MNP with and without hyperthermia, and for comparison between the different MNP based on the expression level. The significance level was set at p ≤0.05.