Innovative Nanopharmaceuticals: Targeting Breast Cancer Stem Cells by Novel Combination of Epigenetic and Anticancer Drugs with Gene Therapy (INNOCENT)

The treatment is based on combining chemotherapy with epigenetics, gene- and radiotherapy into a new nanopharmaceutical – COMBOBOMB. The whole project is in accordance with the safe-by-design principle, where toxicity testing is being performed in parallel with the development of the drug to ensure development of a drug with high efficacy and no serious side effects.

The partners in this project are from Slovakia (coordinator), Greece, Spain, Latvia and Norway (http://www.innocent.pixellaris.com/index.html). Toxicity testing is being performed by the Norwegian partners in close collaboration with the partners developing the nanomaterials. The project partners have selected four cell lines relevant to breast cancer to be applied for toxicity testing, and cell banks have been established. Standard operating procedures (SOPs) have been developed for cultivation of the cells lines and to investigate induction of cell death or DNA damage (cyto- or genotoxicity) by the nanomaterials. A common challenge within nanotoxicology, is interference between the test method and the nanomaterial to be tested. Thus, it is important to apply test methods validated for toxicity testing of nanomaterials.

For testing of cell death (cytotoxicity), several SOPs for different test methods (both colorimetric and label-free) have been worked out: impedance measurements, tryphan blue assay, alamar blue assay (AB) and colony forming efficiency (CFE). One of the drawbacks of traditional laboratory toxicity tests is that they are done under static conditions and do not reflect the dynamics of real life processes. Therefore, UiB have developed an innovative microfluidic device that will mimic better the exposure of breast cancer cells in the body. An advanced 3D in vitro model for breast cancer cells, better reflecting the in vivo situation, is being developed by NILU, and UiB has fabricated a multi-compartment microfluidic chip to support the growth of breast cancer cells, tumor associated stromal cells and microvasculature.

For genotoxicity testing, the modified version of the comet assay is applied to detect DNA strand breaks and specific oxidized DNA base lesions. NILU has modified and validated the comet assay, as well as AB and CFE, for higher throughput measurements and applicability for nanomaterials. Additionally, NILU has established gene mutation tests (Mouse lymphoma assay and HPRT) and test for cancer induction (cell transformation assay; in vitro test for carcinogenicity), and the latter assay has also be modified for higher throughput. UiB and NILU have established the test systems and have tested the received reference gold (Au) nanomaterials on the relevant cellular models for validation of the test models for testing of nanoparticles.

Toxicity of nanomaterials is strongly dependent upon physio-chemical properties, as size. Thus, it is important to analyze size and size distribution of the nanomaterials in the actual dispersion to be applied. SOPs have been established for physiochemical characterization of size and size distribution of nanoparticles in stock as well as in cell culture media by use of nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). Stability of liposome formulations in different dispersion media were analyzed by DLS.

There have been some challenges with designing nanoparticles that are selectively taken up by breast cancer cells and not by normal breast cell. UiB has performed uptake analysis of different liposome (nanocarrier) formulations and with different surface coatings, to detect the most promising candidate for selective uptake in cancer cells and thus targeted delivery. A candidate nanoparticle was selected for further processing, and surface coating modified to obtain increased selective delivery to breast cancer stem cells. Cytotoxicity of the nanocarriers were tested and found not to be cytotoxic by monitoring in real-time via cell-substrate electrical impedance sensing. Work with design and testing of nanocarriers for selective uptake is ongoing.