SUMMARY

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The aim of this project is to elaborate biocompatible silaca coated MNPs with maximized heating power and test them for cancer therapy via magnetic hyperthermia (MH) technique able to induce cell death in vitro. This involves the realization of  the following objectives:

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Objective 1. Silanization of iron oxide MNPs for enhancing their biocompatibility and heating performance:

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The realization of a coating procedure, truly preserving the magnetic integrity of the metallic iron oxide core, represents a necessary step before such iron oxide MNPs with high saturation magnetization could be used in magnetic hyperthermia applications. Since the silica is a versatile material for an additional inorganic outer-layer, we propose to coat theiron oxide MNPs with a silica shell. We strongly believe that this approach will enable to highly improve the biocompatibility of iron oxide MNPs and to reduce their cytotoxic effects. This will allow us to increase the concentration of iron oxide MNPs exposed to cancer cells leading to the internalization of a high amount of iron oxide MNPs inside cells. In addition, this approach will allow to reduce the magnetic dipolar interactions between iron oxide MNPs by increasing the interparticle distances due to the additional silica shell, preserving the excellent heating capabilities of iron oxide MNPs in culture medium also inside cells. The goal is to obtain the optimal thickness of the silica shell for enhancing both the hyperthermia and biocompatibility of iron oxide MNPs.    

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Objective 2. Toxicity and cellular uptake assessment of the silica-coated iron oxide MNPs:

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In general, the toxicology levels of different types of iron oxide MNPs were usually determined by means of only one test - in most cases MTT.  Due to their particular nature and properties, iron oxide MNPs may interfere with the mechanistic aspects of conventional viability assay protocols, producing a false assessment of toxicity.  Therefore, it is also of a paramount importance to carry out additional viability testes in order to properly assess the toxicology level. In accordance with the recent recommendation of the EU NanoSafety Cluster group, we propose in this project to evaluate the in vitro toxicity of silica-coated IOMNPs by using four complementary types of viability assays: the membrane integrity test by measuring the release of lactate dehydrogenase (LDH), the metabolic activity by the Alamar blue test and the quantitation of the ATP, and the Trypan Blue test for the viability assay. The goal is to establish the concentration threshold of silica-coated iron oxide MNPs corresponding to their intrinsic toxicity and to determine the maximum load per cell.  The interactions of silica-coated iron oxide MNPs with cells, the mechanisms involved in their internalization in cells as well as the fate of them inside the cells will be also investigated.

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Objective 3. Demonstration of magnetic hyperthermia capabilities of silica coated iron oxide MNPs to induce apoptosis:

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Cancer cells incubated with silica-coated iron oxide MNPs at concentrations below their intrinsic toxicity level will be placed inside a coil and will be exposed to external AC magnetic fields. The duration of the exposure will be varied while the H*f factor will be kept below the safety limit of MH.  A systematic study based on above the mentioned viability tests of the hyperthermia effect generated by silica-coated iron oxide MNPs inside cells will be performed.  The final goal is to demonstrate the capability of silica-coated iron oxide MNPs to induce in vitro apoptosis of the cancer cells at concentration below their intrinsic toxicity and in AC magnetic fields satisfying the condition H*f<5x10^9 A/ms. Moreover, there are still many open questions related to the mechanisms involved in the magnetic hyperthermia-induced cell apoptosis. In this regard analysis of the cell structures after MH will be employed.