Dextran

This measure is based on the intestinal permeability towards 4000 Da fluorescent dextran–FITC (DX-4000–FITC) (FD4000; Sigma-Aldrich, St. Louis, Missouri, USA) as described.6 (link) 43 (link) Briefly, mice that had fasted for 6 h were given DX-4000–FITC by gavage (500 mg/kg body weight, 125 mg/ml). After 1 h and 4 h, 120 μl of blood was collected from the tip of the tail vein. The blood was centrifuged at 4°C, 12 000 g for 3 min. Plasma was diluted in an equal volume of PBS (pH 7.4) and analysed for DX-4000–FITC concentration with a fluorescence spectrophotometer (HTS-7000 Plus-plate-reader; Perkin Elmer, Wellesley, Massachusetts, USA) at an excitation wavelength of 485 nm and emission wavelength of 535 nm. Standard curves were obtained by diluting FITC–dextran in non-treated plasma diluted with PBS (1:3 v/v).

As previously mentioned, MFH is a potential application of our instrument. Magnetic nanoparticles (MNPs) are required to dissipate enough heat to raise the temperature of the tumor up to 41 °C–47 °C, in presence of an AMF. Synomag^®-D (Micromod, Rostock, Germany) MNPs with PEG 25000-OMe groups were used in this work (Lot # 06021104–01). These heat mediators are core–shell particles with a nanoflower maghemite core, covered by a dextran shell and functionalized with PEG-OMe. The MNPs are also suitable for MFH applications and have a prolonged circulation time in blood. Here, their magneto-physical properties are briefly described. The MNP hydrodynamic diameter was determined with the aid of dynamic light scattering (DLS) using the NanoBrook Omni particle size analyzer (Brookhaven Instruments Corporation, Long Island, NY, USA). Here, the MNPs were suspended in RPMI cell culture media from 0 to 72 h and measurements were taken every 24 h. The MNPs maintained their 70 nm hydrodynamic diameter size, meaning that they were not aggregating over time (see figure S8 in subsection D of the Supplementary Information). This was in accordance with the certificate of analysis from the company. The specific absorption rate (SAR) of the MNPs was 891.25 ± 21.66 W/g_Fe and was determined using the slope of the increasing temperature profiles of nanoparticles when exposed to the alternating magnetic field (28.33 kA m⁻¹ at 258 kHz) of an EasyHeat 8310LI induction heater (Ambrell Corporation, Rochester, NY, USA). Iron quantification was conducted using an Infinite 200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The stock MNPs were 6.20 mg_Fe ml⁻¹ when received and 5.99 mg_Fe ml⁻¹ after filtration.

HESCs were grown in DMEM/F12 medium without phenol red (Sigma-Aldrich, St. Louis, MO, United States of America), added with 10% charcoal dextran-treated fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, United States of America), 1% ITS - Premix (BD Biosciences, San Jose, CA, United States of America) and 5 ng/mL puromycin (Thermo Fisher Scientific, Ottawa, ON, CAN). Every culture was kept at 37 °C in an incubator with 5% CO₂. HESCs were cultivated for a day after being seeded at a density of 4 × 10⁵ cells per plate in 60-mm tissue culture dishes with full culture media. HESCs were treated with BMP2 (0, 10, 25, or 50 ng/ml) for the concentration-dependent research or with 25 ng/mL BMP2 for the time-course study after serum deprivation in DMEM/F12 media without FBS for 18 h. For the concentration-dependent investigation, cells were taken at 24 h, while for the time-course study, cells were taken at 3, 6, 12, 24 h, and 48 h.