*3.3. Stability*

Like in many other applications, the use of magnetic nanoparticles in health applications requires to ensure su fficient stability. Previous results from our laboratory demonstrated that, specifically when it comes to hyperthermia applications, the stability, either electrostatically or polymerically achieved, is an essential factor for optimizing the performance of the system [22]. Hence, in this part of the study, we focus on the possibility of improving hyperthermia by favoring stability. It must be recalled that superparamagnetic nanoparticles interact attractively through van der Waals interactions and dipolar magnetic ones (if remanence is not negligible, which is not our case at room temperature, see Figure 3). Furthermore, the repulsive interactions due to the surface charge will be very low at physiological pH values in the case of MNPs, and larger in the case of BMNPs (Figure 5). In addition, steric hindrance could contribute to the stability of the biomimetic nanoparticles through the presence of surface MamC molecules. In order to confirm this possibility, we evaluated the sedimentation behavior vs. time of the samples (pure end members and mixtures of BMNPs and MNPs). The time evolution of the boundary between sedimented volume (height *h*) and clear supernatant, relative to the initial height *h*0, will be used as a simple test of stability. Data are plotted in Figure 6. Surprisingly, it can be observed that, although the stability of bare MNPs is lower than that of BMNPs, as expected from the steric and electrostatic repulsions due to the MamC coating, adding BMNPs to the former at a relative concentration of 25/75 (25 B + 75 M) brings about a measurable increase of stability. This may be the result of a compromise between the smaller size of MNPs favoring stability and the addition of the protective coating represented by the BMNPs. As a result, an ideal system with a mixed composition emerges.

**Figure 6.** Height of the sedimented volume relative to its initial value as a function of time for MNPs (-), BMNPs ( -), 60 B + 40 M (), and 25 B + 75 M (). Inset: Short-time detail.

### *3.4. Performance in Hyperthermia*

As a novel field of application of the two kinds of particles and their mixtures, we consider to what extent they are e fficient magnetic hyperthermia agents. Figure 7 shows the time evolution of the temperature of the suspensions of MNPs, BMNPs and the two mixtures 25 B + 75 M and 60 B + 40 M under the influence of an alternating magnetic field at the indicated frequencies and a fixed magnetic field strength of 18 kA/m. All types of nanoparticles are able to raise the temperature, the fastest rise occurring for the highest frequency. This effect is particularly visible at longer times. The rate of temperature increase is maximum (~34 ◦C/min) in mixture 25 B + 75 M and minimum in BMNPs (~17 ◦C/min) at the highest frequency. It appears that inorganic MNPs are better hyperthermia agents than BMNPs. This justifies their presence in the mixtures under study. They make it possible to design a composition of nanoparticles that (i) can be guided to the target by the application of an external magnetic field; (ii) behave as suitable drug nanocarriers, stable at physiological pH values and from which the drug release is dependent of an external stimuli, e.g., acidic tumor environment (these are the BMNPs); (iii) produce a fast increase of the temperature of the system upon the application of an external magnetic field (these are the MNPs).

**Figure 7.** Time evolution of the temperature of the MNP suspensions, for different frequencies: 197 kHz (-), 236 kHz (), and 280 kHz (). Field strength: *H*0 = 18 kA/m. Sample volume 0.5 mL; particle concentration: 25 mg/mL.

In fact, maximum *SAR* (up to 96.2 W/g) and *ILP* (1.26 nHm2kg−1) values were obtained for the 25 B + 75 M sample, followed by MNPs, 60 B + 40 M and, finally, BMNPs (Figure 8, Table 1). The lower heating induced by BMNPs is probably related to the larger size of the particles and to the presence of MamC attached to their surface, which probably hinders the rotation of the nanoparticles. The increased *SAR* and *ILP* values obtained for the 25 B + 75 M samples is promoted by stability, as single particles with close-to-spherical symmetry rotate under the action of the field less impeded than in the case of aggregated particles. In addition, it is likely that the field frequencies used are closer to those of maximum phase lag between magnetization and field, for the MNPs than for the BMNPs, so the latter would play here the role of favoring stability.

**Figure 8.** Frequency dependence of (**a**) *SAR* and (**b**) *ILP*, for the investigated systems. MNPs (-), BMNPs (-), 60 B + 40 M (), and 25 B + 75 M (). Magnetic field strength: 18 kA/m; particle concentration: 25 mg/mL.

**Table 1.** Summary of Specific Absorption Rate (SAR), Intrinsic Loss Power (ILP) calculations, and temperature increase after 60 s exposition time, Δ*T*, for the different samples tested [inorganic Magnetic Nanoparticles (MNPs) and MamC-medianted Biomimetic Magnetic Nanoparticles (BMNPs)], and the field frequencies indicated.

