## 2 Materials and Methods
### Methods
#### 2.2.4 Drug Release Behavior  
In vitro drug release behavior of doxorubicin-loaded PNIPAM/magnetite nanoparticles was investigated at 7.45 pH and 37 \({}^{\circ}\)C. Briefly, 5 mL of doxorubicin-loaded nanoparticles in phosphate-buffered saline solution (PBS, pH 7.45) was placed in a tubular cellulose membrane, followed by immersion in flasks with a fixed volume (35 mL). The flasks were further incubated in an orbital mixer (Benchmark Scientific, Sayreville, NJ, USA) at 400 rpm, and 37.0 \(\pm\) 0.5 \({}^{\circ}\)C. A total of 5 mL of PBS dialysate was collected at predetermined time intervals and then investigated by UV-VIS spectroscopy (SHIMADZU UV-3600 instrument). To maintain a constant volume, after each collection, 5 mL of fresh PBS was added to every flask. Release and encapsulation efficiencies were computed as follows (Equations (1) and (2)):  
\[\text{RE }(\%)=\frac{\text{amount of released DOX}}{\text{amount of the loaded DOX}}\times 100 \tag{1}\]  
\[\text{EE }(\%)=\frac{\text{amount of the loaded DOX}-\text{amount of unloaded DOX}}{\text{amount of the loaded DOX}}\times 100 \tag{2}\]  
.2.5 In Vitro Biological Investigation on Free and Doxorubicin-Loaded PNIPAM/Magnetite Nanoparticles  
Cell Culture Model  
Pristine and doxorubicin-loaded PNIPAM (0.25%)/magnetite NPs were subjected to in vitro biological evaluation in terms of determining their efficacy on cancer cells (MCF7 human breast adenocarcinoma cell line, ATCC). In this view, the cells were propagated in standard conditions of culture (37 \({}^{\circ}\)C, humidified atmosphere, and 5% CO\({}_{2}\)) in complete culture medium (DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin mixture). MTT Assay  
To determine the NPs' working dose (IC50-inhibitory concentration 50), MCF7 cells were seeded in flat bottom 96-well plates at an initial cell density of 104 cells/cm\({}^{2}\) and treated after 24 h with the following dilutions of pristine and DOX-loaded PNIPAM/mag NPs: 25 mg/mL, 20 mg/mL, 15 mg/mL, 10 mg/mL, 7.5 mg/mL, 5 mg/mL, 1 mg/mL, 500 \(\mu\)g/mL, 250 \(\mu\)g/mL, 100 \(\mu\)g/mL, and 50 \(\mu\)g/mL. The treatments were kept in contact with the cells for 24 h, and then the monolayers were washed and incubated for 4 h with 1 mg/mL MTT solution. In the end, the MTT solution was washed, and the formazan crystals formed in the metabolically active cells were dissolved using isopropanol. The absorbance of the resulting solutions was determined at 550 nm using FlexStation III multimodal reader (Molecular Devices, San Jose, CA, USA). Untreated samples were prepared as controls. Fluorescence Microscopy Assay  
To investigate cells morphology under exposure to pristine and doxorubicin-loaded PNIPAM/mag NPs, MCF7 cells were seeded in 12-well plates at an initial density of 104 cells/cm\({}^{2}\) and after 24 h were exposed for one day to 100 \(\mu\)g/mL NPs. The monolayers were washed with PBS and fixed for 20 min with paraformaldehyde(PFA) 4% at 24 h of treatment. The cell membranes were then permeabilized for 30 min with a solution of bovine serum albumin (BSA) 2% and Triton \(\times\)100 (0.1%), and the cytoskeleton's actin filaments were stained for 1 h and 30 min at 37 \({}^{\circ}\)C and in darkness with a solution of Phalloidin-FITC. In the end, the staining solution was discarded, and the cell nuclei were stained for 10 min with DAPI solution. An untreated monolayer was prepared similarly and served as control. All the samples were investigated by fluorescence microscopy using an Olympus IX73 inverted microscope with epifluorescence modulus (Olympus, Tokyo, Japan), and images were captured and processed using CellSense F software, version 1.11. Statistical Analysis  
The graphical representation of the obtained results was performed using the Graph-Pad Prism software, version 5.0. The mean of the obtained data was obtained from three independent experiments and is presented as the arithmetic mean S.D.