Fluorescent dye doped core-shell silica nonoparticles for diagnostic and therapeutic applications: preliminary studies as innovative multimodal tool in imaging and therapy

Since most biologically active macromolecules are natural nanostructures, operating in the same scale of biomolecules gives the great advantage to enhance the interaction with cellular components, as cell membrane and proteins. Among the variety of new nanomaterials investigated and developed during last decades, nanoparticles (NPs) provide a particularly useful platform, showing unique properties with wide-ranging applications. As a result, NPs are by far the most versatile and deeply studied class of nanomaterials. NTB700 NPs, employed for our research, are fluorescent core-shell silica nanoparticles, synthesized through a micelle-assisted method. The aim of the present study was firstly NP characterization and interaction with some biological models (U937 and peripheral blood mononuclear cells), describing the specific triggered biologic response, in order to exploit this new technology for future applications. The use of U937 cells guaranteed the possibility to collect data on a homogeneous, numerically plentiful, myeloid population, whereas peripheral blood mononuclear cells (PBMCs) allowed for the simultaneous analysis of NP effects on many and varied important immune cells like B-cells, T-cells, monocytes and natural killer cells, which is fundamental once introduced into the blood torrent. Initially, we investigated NTB700 physicochemical properties, their size and the presence of aggregates, related to pH. For the purpose of using these nanomaterials as drug delivery and/or imaging tools, it is necessary to study their endocytosis, sub-cellular fate, localization and clearance mechanisms in target cells. To characterize how NTB700 NPs were taken up by cells, we quantified their incorporation through flow cytometry, supported by a qualitative analysis performed by confocal microscopy. Results showed that the internalization process is time-, concentration-, energy-and cell type-dependent. Once inside the cells NPs were transported within plasma-membrane bounded vesicles along microtubules to organelles, as lysosomes and, in particular, mitochondria, but without affecting cell viability. Cells, in fact, did not go to death but we may hypothesize a slight arrest of cell growth. By flow cytometry and confocal microscopy, we also investigated the effect of NTB700 NPs on several trafficking routes and pathways involving e.g. lysosomes, autophagosome, mitochondria and ROS production. Based on previous results, we isolated mitochondria from U937 cells treated with NPs to more in deep investigate their localization and to study the mitochondrial redox environment, which is important for mitochondrial dysfunction and cell death. Finally, both lymphoid and myeloid cells were able to release NPs and this is essential to assess their biosafety and future developments. Since NPs appeared a promising imaging platform, showing a specific subcellular localization, we decided to conjugate one of the most commonly used anticancer drug, doxorubicin (DOX). We tested these modified NPs on breast cancer cell line MCF-7, since DOX is currently one of the most effective agents in the treatment of breast cancer. We evaluated DOX-NP cytotoxicity and effect on the expression of CD44 antigen, a molecule involved in adhesion, therefore in cell spreading in tumour invasion, both on cell surface and on extracellular vesicles (EVs) released from cells, compared to free DOX and stand-alone NPs. We did not observe an increased cytotoxic effect, as we expected, but an interesting ability to release a minor amount of CD44+ EVs in cells treated with DOX-NPs. In parallel, we also conjugated some of the most common monoclonal antibodies (mAbs) on NP surface, by exploiting the amine groups on NP shell, to investigate the targeting abilities of NTB700 NPs. Among the various potential applications of this versatile technology, we decided to study in deep the capability of multiple dye doped NPs to be fluorescent probes. NPs resulted brilliant probes for these immune cell antigens, confirming their capability to target the desired cells. All these data allow us to consider fluorescent SiNPs a promising platform for a multifunctional device in which collimate both the imaging and therapeutic applications in a unique tool conveying to a specific target.