## 4 Discussion  
The local surrounding (e.g., environment) of nanoparticles and nanoparticles hierarchical organization may have an impact on different therapeutic [17] and diagnostic modalities [35]. The mechanical properties of the surrounding environment [36] or the clustering SPIONs may thus affect the nanoparticles' heating yields [17]. In magnetic hyperthermia, external mechanical constraints [36], as well as nanoparticle confinement or aggregation, decreases the thermal yield because mechanically constrained or aggregated nanoparticles have less freedom to rotate (which inhibits Brownian relaxation) or because the inter-nanoparticle distance decreases, increasing nanoparticle interactions (which hampers Neel relaxation). Upon cellular internalization, nanoparticles become confined in endosomes, and one is given as an example in Figure 4. The net result of nanoparticle confinement/aggregation is diminished heat generation in magnetic hyperthermia. Conversely, studies performed in living cells showed that, in photothermala modality, the heating does not decline upon nanoparticle internalization in cells. Nanoparticle enclosures within endosomes do not decrease the nanoparticles' capacity to heat. Importantly, intracellular aggregation showed a small increase of the nanoparticles' photothermal heating [17,27]. Based on these findings, we herein investigated whether well-defined SPION clustering, and encapsulation by a silica shell (Figure 1), might affect the photothermal yield. In this context, we tested the heating potential of individual SPIONs, individual silica-encapsulated SPIONs (SPIONs-SIL), and silica-encapsulated SPION clusters (MNCs). These MNC assemblies highly resembled endosomes (Figure 4), which are cellular compartments in which nanoparticles are circumscribed upon nanoparticle endocytosis by cells. Figure 4: Representative transmission electron micrograph exemplifying an endosome within the cytoplasm of a cell. M denotes a mitochondrion in the vicinity of the endosome, which is laden with magnetic nanoparticles. Structurally, the nanoparticles within endosomes resemble MNCs. In the present study, we compared two heating modalities: photothermal and magnetic hyperthermia. We aimed to provide a better understanding of the outcome of nanoparticle heating behaviors in relation to their configurations (non-encapsulated vs. encapsulated and individual vs. clustered). Indeed, all compared nanoparticle types consisted of identical maghemite core nanocrystals [32]. In photothermal, at the iron concentration of 30 mM, SPIONs-SIL heated to the highest extent (45.7 \({}^{\circ}\)C), followed by SPIONs (43.5 \({}^{\circ}\)C). Here, we first emphasize that the laser power was clinically relevant (0.3 W/cm\({}^{2}\)), and second, the iron concentration was much lower than the one that is used for magnetic hyperthermia (Figure 3A). The comparison of SPIONs and SPIONs-SIL indicates that the encapsulation with silica leads to a better heating yield in this heating modality. As the nanoparticles compared in this study consist of maghemite, a potentially different crystal phase (e.g., magnetite) cannot explain the difference in nanoparticle heating. The most obvious cause for heating enhancement is thus the silica coating. The presence of nanoparticles may increase the thermal conductivity of fluids [37], and studies show that silica nanoparticles can increase the critical heat flux [38]. Moreover, studies showed that, in water suspensions, silica-coated gold nanoparticles dissipated heat much faster than bare (citrate-stabilized) gold nanoparticles [39]. In line with these findings observed for gold nanoparticles [39], our silica-coated SPIONs also dissipated heat to a greater extent than non-encapsulated SPIONs. In contrast, the clustering of SPIONs into well-defined, endosome-like structures (Figure 4) did not enhance photothermal heating. As MNCs are larger than SPIONs-SIL, they might dissipate heat at a slower rate [40].