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Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles' integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

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Gold nanoparticles can act as photothermal agents to generate local tumor heating and subsequent depletion upon laser exposure. Herein, photothermal heating of four gold nanoparticles and the resulting induced cancer cell death are systematically assessed, within extra- or intracellular localizations. Two state-of-the-art gold nanorods are compared with small nanospheres (single-core) and nanoraspberries (multicore). Heat generation is measured in water dispersion and in cancer cells, using lasers at wavelengths of 680, 808, and 1064 nm, covering the entire range used in photothermal therapy, defined as near infrared first (NIR-I) and second (NIR-II) windows, with NIR-II offering more tissue penetration. When dispersed in water, gold nanospheres provide no significant heating, gold nanorods are efficient in NIR-I, and only gold nanoraspberries are still heating in NIR-II. However, in cells, due to endosomal confinement, all nanoparticles present an absorption red-shift translating visible and NIR-I absorbing nanoparticles into effective NIR-I and NIR-II nanoheaters, respectively. The gold nanorods then become competitive with the multicore nanoparticles (nanoraspberries) in NIR-II. Similarly, once in cells, gold nanospheres can be envisaged for NIR-I heating. Remarkably, nanoraspberries are efficient nanoheaters, whatever the laser applied, and the extra- versus intra-cellular localization demonstrates treatment versatility.

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Cancerous cells and the tumor microenvironment are among key elements involved in cancer development, progression, and resistance to treatment. In order to tackle the cells and the extracellular matrix, we herein propose the use of a class of silica-coated iron oxide nanochains, which have superior magnetic responsiveness and can act as efficient photothermal agents. When internalized by different cancer cell lines and normal (non-cancerous) cells, the nanochains are not toxic, as assessed on 2D and 3D cell culture models. Yet, upon irradiation with near infrared light, the nanochains become efficient cytotoxic photothermal agents. Besides, not only do they generate hyperthermia, which effectively eradicates tumor cells in vitro, but they also locally melt the collagen matrix, as we evidence in real-time, using engineered cell sheets with self-secreted extracellular matrix. By simultaneously acting as physical (magnetic and photothermal) effectors and chemical delivery systems, the nanochain-based platforms offer original multimodal possibilities for prospective cancer treatment, affecting both the cells and the extracellular matrix.

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Magnetic nanoparticles (MNPs) internalized within stem cells have paved the way for remote magnetic cell manipulation and imaging in regenerative medicine. A full understanding of their interactions with stem cells and of their fate in the intracellular environment is then required, in particular with respect to their surface coatings. Here, we investigated the biological interactions of MNPs composed of an identical magnetic core but coated with different molecules: phosphonoacetic acid, polyethylene glycol phosphonic carboxylic acid, caffeic acid, citric acid, and polyacrylic acid. These coatings vary in the nature of the chelating function, the number of binding sites, and the presence or absence of a polymer. The nanoparticle magnetism was systematically used as an indicator of their internalization within human stem cells and of their structural long-term biodegradation in a 3D stem cell spheroid model. Overall, we evidence that the coating impacts the aggregation status of the nanoparticles and subsequently their uptake within stem cells, but it has little effect on their intracellular degradation. Only a high number of chelating functions (polyacrylic acid) had a significant protective effect. Interestingly, when the nanoparticles aggregated prior to cellular internalization, less degradation was also observed. Finally, for all coatings, a robust dose-dependent intracellular degradation rate was demonstrated, with higher doses of internalized nanoparticles leading to a lower degradation extent.

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Gold nanoraspberries were synthesized by a seed-mediated synthesis with polyethylene glycol-functionalized bisphosphonates. The original structure shifted the optical absorption to infrared, revealing very efficient photothermal properties within the 2nd biological transparency window and leading to cancer cell necrosis at moderate intracellular doses and low (safe) laser power.

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While magnetic nanoparticles offer exciting possibilities for stem cell imaging or tissue bioengineering, their long-term intracellular fate remains to be fully documented. Besides, it appears that magnetic nanoparticles can occur naturally in human cells, but their origin and potentially endogenous synthesis still need further understanding. In an effort to explore the life cycle of magnetic nanoparticles, we investigated their transformations upon internalization in mesenchymal stem cells and as a function of the cells' differentiation status (undifferentiated, or undergoing adipogenesis, osteogenesis, and chondrogenesis). Using magnetism as a fingerprint of the transformation process, we evidenced an important degradation of the nanoparticles during chondrogenesis. For the other pathways, stem cells were remarkably "remagnetized" after degradation of nanoparticles. This remagnetization phenomenon is the direct demonstration of a possible neosynthesis of magnetic nanoparticles in cellulo and could lay some foundation to understand the presence of magnetic crystals in human cells. The neosynthesis was shown to take place within the endosomes and to involve the H-subunit of ferritin. Moreover, it appeared to be the key process to avoid long-term cytotoxicity (impact on differentiation) related to high doses of magnetic nanoparticles within stem cells.

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A gold therapeutic nanoplatform with the same molecule used as reductant, coating and therapeutic agent has been developed in a one-pot, one-phase process using alendronate, a drug from the bisphosphonate family known for its antitumor effects. In addition, the core made of gold nanoparticles (NPs) brings thermal functionalities under irradiation within the first biological window (650-900 nm). The Au@alendronate nanoplatform thus provided a combined antitumor activity through drug delivery and photothermal therapy. Au@alendronate NPs inhibited in vitro the proliferation of prostate cancer cells (PC3) in a dose-dependent manner, with an IC50 value of 100 µM. Under NIR irradiation a temperature increase was observed leading to a reduction of the IC50 value to 1 µM, with total tumor cell death at 100 µM.

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Providing appropriate means for heat generation by low intratumoral nanoparticle concentrations is a major challenge for cancer nanotherapy. Here we propose RGD-tagged magnetosomes (magnetosomes@RGD) as a biogenic, genetically engineered, inorganic platform for multivalent thermal cancer treatment. Magnetosomes@RGD are biomagnetite nanoparticles synthesized by genetically modified magnetotactic bacteria thanks to a translational fusion of the RGD peptide with the magnetosomal protein MamC. Magnetosomes@RGD thus combine the high crystallinity of their magnetite core with efficient surface functionalization. The specific affinity of RGD was first quantified by single-cell magnetophoresis with a variety of cell types, including immune, muscle, endothelial, stem and cancer cells. The highest affinity and cellular uptake was observed with PC3 prostatic and HeLa uterine cancer cells. The efficiency of photothermia and magnetic hyperthermia was then compared on PC3 cells. Unexpectedly, photothermia was far more efficient than magnetic hyperthermia, which was almost totally inhibited by the cellular environment. RGD targeting was then assessed in vivo at tumor site, in mice bearing PC3 tumors. As a result, we demonstrate that targeted magnetic nanoparticles could generate heat on a therapeutic level after systemic administration, but only under laser excitation, and successfully inhibit tumor progression.

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