Progress in the design of nanoscale magnets for localized hyperthermia cancer therapy has been largely driven by trial-and-error approaches, for instance, by changing of the stoichiometry composition, size, and shape of the magnetic entities. So far, widely different and often conflicting heat dissipation results have been reported, particularly as a function of the nanoparticle concentration. Thus, achieving hyperthermia-efficient magnetic ferrofluids remains an outstanding challenge. Here we demonstrate that diverging heat-dissipation patterns found in the literature can be actually described by a single picture accounting for both the intrinsic magnetic features of the particles (anisotropy, magnetization) and experimental conditions (concentration, magnetic field). Importantly, this general magnetic-hyperthermia scenario also predicts a novel non-monotonic concentration dependence with optimum heating features, which we experimentally confirmed in iron oxide nanoparticle ferrofluids by fine-tuning the particle size. Overall, our approach implies a magnetic hyperthermia trilemma that may constitute a simple strategy for development of magnetic nanomaterials for optimal hyperthermia efficiency.

Magnetic nanoparticles exposed to alternating magnetic fields have shown a great potential acting as magnetic hyperthermia mediators for cancer treatment. However, a dramatic and unexplained reduction of the nanoparticle magnetic heating efficiency has been evidenced when nanoparticles are located inside cells or tissues. Recent studies suggest the enhancement of nanoparticle clustering and/or immobilization after interaction with cells as possible causes, although a quantitative description of the influence of biological matrices on the magnetic response of magnetic nanoparticles under AC magnetic fields is still lacking. Here, we studied the effect of cell internalization on the dynamical magnetic response of iron oxide nanoparticles (IONPs). AC magnetometry and magnetic susceptibility measurements of two magnetic core sizes (11 and 21 nm) underscored differences in the dynamical magnetic response following cell uptake with effects more pronounced for larger sizes. Two methodologies have been employed for experimentally determining the magnetic heat losses of magnetic nanoparticles inside live cells without risking their viability as well as the suitability of magnetic nanostructures for in vitro hyperthermia studies. Our experimental results-supported by theoretical calculations-reveal that the enhancement of intracellular IONP clustering mainly drives the cell internalization effects rather than intracellular IONP immobilization. Understanding the effects related to the nanoparticle transit into live cells on their magnetic response will allow the design of nanostructures containing magnetic nanoparticles whose dynamical magnetic response will remain invariable in any biological environments, allowing sustained and predictable in vivo heating efficiency.

Iron oxide nanoparticles have found an increasing number of biomedical applications as sensing or trapping platforms and therapeutic and/or diagnostic agents. Most of these applications are based on their magnetic properties, which may vary depending on the nanoparticle aggregation state and/or concentration. In this work, we assess the effect of the inter- and intra-aggregate magnetic dipolar interactions on the heat dissipation power and AC hysteresis loops upon increasing the nanoparticle concentration and the hydrodynamic aggregate size. We observe different effects produced by inter- (long distance) and intra-aggregate (short distance) interactions, resulting in magnetizing and demagnetizing effects, respectively. Consequently, the heat dissipation power under alternating magnetic fields strongly reflects such different interacting phenomena. The intra-aggregate interaction results were successfully modeled by numerical simulations. A better understanding of magnetic dipolar interactions is mandatory for achieving a reliable magnetic hyperthermia response when nanoparticles are located into biological matrices.

In the pursuit of controlling the heat exposure mediated by magnetic nanoparticles, we provide new guidelines for tailoring magnetic relaxation processes via dipolar interactions. For this purpose, highly crystalline and monodisperse magnetic iron oxide nanocrystals whose sizes range from 7 to 22 nm were synthesized by thermal decomposition of iron organic precursors in 1-octadecene. The as-synthesized nanoparticles are soft nanomagnets, showing superparamagnetic-like behavior and SAR values which progressively increase with particle size, field frequency, and amplitude up to 3.6 kW/g Fe . Our data show the influence of media viscosity, particle size, and concentration on dipolar interactions and consequently on the magnetic relaxation processes related to the heat release. Understanding the role of dipolar interactions is of great importance toward the use of iron oxide nanoparticles as efficient hyperthermia mediators.

Hysteresis losses in magnetic nanoparticles constitute the basis of magnetic hyperthermia for delivering a local thermal stress. Nevertheless, this therapeutic modality is only to be realised through a careful appraisal of the best possible intrinsic and extrinsic conditions to the nanoparticles for which they maximise and preserve their heating capabilities. Low frequency (100 kHz) hysteresis loops accurately probe the dynamical magnetic response of magnetic nanoparticles in a more reliable manner than calorimetry measurements, providing conclusive quantitative data under different experimental conditions. We consider here a set of iron oxide or cobalt ferrite nanocubes of different sizes, through which we experimentally and theoretically study the influence of the viscosity of the medium on the low frequency hysteresis loops of magnetic colloids, and hence their ability to produce and dissipate heat to the surroundings. We analyse the role of nanoparticle size, size distribution, chemical composition, and field intensity in making the magnetisation dynamics sensitive to viscosity. Numerical simulations using the stochastic Landau-Lifshitz-Gilbert equation model the experimental observations in excellent agreement. These results represent an important contribution towards predicting viscosity effects and hence to maximise heat dissipation from magnetic nanoparticles regardless of the environment.

Herein, by studying a stepwise phase transformation of 23 nm FeO-Fe3O4 core-shell nanocubes into Fe3O4, we identify a composition at which the magnetic heating performance of the nanocubes is not affected by the medium viscosity and aggregation. Structural and magnetic characterizations reveal the transformation of the FeO-Fe3O4 nanocubes from having stoichiometric phase compositions into Fe 2+ deficient Fe3O4 phases. The resultant nanocubes contain tiny compressed and randomly distributed FeO sub-domains as well as structural defects. This phase transformation causes a tenfold increase in the magnetic losses of the nanocubes, which remains exceptionally insensitive to the medium viscosity as well as aggregation unlike similarly sized single-phase magnetite nanocubes. We observe that the dominant relaxation mechanism switches from Néel in fresh core-shell nanocubes to Brownian in partially oxidized nanocubes and once again to Néel in completely treated nanocubes. The Fe 2+ deficiencies and structural defects appear to reduce the magnetic energy barrier and anisotropy field, thereby driving the overall relaxation into Néel process. The magnetic losses of the particles remain unchanged through a progressive internalization/association to ovarian cancer cells. Moreover, the particles induce a significant cell death after being exposed to hyperthermia treatment. Here, we present the largest heating performance that has been reported to date for 23 nm iron oxide nanoparticles under cellular and intracellular conditions. Our findings clearly demonstrate the positive impacts of the Fe 2+ deficiencies and structural defects in the Fe3O4 structure on the heating performance under cellular and intracellular conditions.

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D 1 receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the ␣ isoform of GRK4; the ␤, ␥, and ␦ isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4␣ is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D 1 receptor by GRK4␣ results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number-all of which are abolished in a D 1 receptor mutant containing T428V and S431A. The increase in internalized D 1 receptors induced by GRK4␣ phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D 1 receptor by GRK4␣ does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4␣. These observations shift the attenuation of D 1 receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization. Dopamine signaling in mammals is mediated by five G protein-coupled receptor (GPCR) proteins divided into two groups based upon sequence homology, G protein coupling, signaling pathways, pharmacological profiles, and desensitization kinetics (Sibley and Monsma, 1992;Missale et al., 1998 Upon agonist activation, GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response under continued agonist stimulation (Ferguson et al., 1996;Gainetdinov et al., 2004). Desensitization involves phosphorylation of the receptor by GRKs and/or second messenger-activated kinases (cAMP-dependent protein kinase or protein kinase C). Homologous desensitization of GPCRs involves only activated receptors and is primarily mediated by GRKs. GRKs are serine/threonine-directed protein kinases composed of seven isoforms divided into three families (Penela et al., 2003). GRK1 and GRK7 compose the rhodopsin kinase/visual family, are expressed exclusively in retina, and participate in desensitization of opsins in rods and cones (Somers and Klein, 1984;Hisatomi et al., 1998;Weiss et al., 1998). GRK2 (␤ARK1) and GRK3 (␤ARK2) were originally identified as regulating the ␤-adrenergic receptor and com- ABBREVIATIONS: GPCR, G protein-coupled receptor; ␤ARK, ␤-adrenergic ...

Homologous desensitization of D 1 dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D 1 receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.