ROMANIAN CONTRIBUTION | EURONANOMED-THERAGET

The project aims at validating the innovative design of targeting multimodal imaging (MRI, SPECT &PET) and therapeutic (magnetic hyperthermia - MH) multifunctional nanoplatforms (NPFs) using already validated targeting ligands (TLs) for tumors. NPFs, that will be developed, will allow multimodal imaging and therapy, monitoring the effect of treatment by imaging.

The project consortium proposes i) engineering of two types of DNPs (one for diagnosis and one combining diagnosis and therapy); ii) the functionalization of DNPs with TLs and/or SPECT/PET probes and iii) preclinical proof-of-concept for improved diagnostics and/or therapy of solid tumors.

As a member of the consortium, the Romanian partner is involved in experiments related to the in vitro and in vivo cytotoxicity assays, MH in vitro, and in vivo efficacy of NPFs.

The cytotoxicity of NPFs will be studied in vitro, on breast cancer (MCF-7 and MDA-MB-231) and ovarian cancer (SKOV3 and SW626) cell lines, as well as in vivo. The cells will be analyzed to check the expression of apoptotic signature and the plasma membrane integrity. The intrinsic and comparative cytotoxic properties of NPFs will be assessed by using 3 standardized tests: the membrane integrity test by measuring the release of lactate dehydrogenase (LDH) while the metabolic activity by the Alamar blue test and Neutral Red test aiming at establishing the concentration threshold of NPFs corresponding to their intrinsic toxicity.

The efficiency of the MH treatment on normal/cancer cells loaded with different amounts of NPFs will be evaluated and the optimal MH parameters (frequency and magnetic field amplitude and the number of MH cycles) will also be determined. The goal is to demonstrate the capability of DNPs to induce in vitro apoptosis of the cancer cells at concentrations below their intrinsic toxicity and in AC magnetic fields satisfying the condition Hxf<5x109 Am-1s-1. TEM analysis of the cell structures after MH would answer many open questions related to the mechanisms involved in the magnetic hyperthermia-induced cell apoptosis.

Before in vivo MH, the safety in vivo profile of the DNPs will be determined on BALB/c mice by the assessment of the acute toxicity after a single intravenous administration as well as the subacute toxicity after repeated daily intravenous administration during 14 or 28 days. Moreover, the biodistribution of the DNPs will be assessed after the animals will be sacrificed and compared with the imaging data. The in vivo therapeutic capabilities of DNPs will be assessed on mice having induced subcutaneous breast cancer in a cell-derived xenograft (CDX) model by exposing the mice treated with 20 nm DNPs to alternating magnetic fields (AMF) different field amplitudes and at several frequencies following the tumor volume evolution under MH.