No abstract

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Cell death triggered by photodynamic therapy can occur through different mechanisms: apoptosis, necrosis or autophagy. However, recent studies have demonstrated the existence of other mechanisms with characteristics of both necrosis and apoptosis. These new cell death pathways, collectively termed regulated necrosis, include a variety of processes triggered by different stimuli. In this study, we evaluated the cell death mechanism induced by photodynamic treatments with two photosensitizers, meso-tetrakis (4-carboxyphenyl) porphyrin sodium salt (Na-H2TCPP) and its zinc derivative Na-ZnTCPP, in two human breast epithelial cell lines, a non-tumoral (MCF-10A) and a tumoral one (SKBR-3). Viability assays showed that photodynamic treatments with both photosensitizers induced a reduction in cell viability in a concentration-dependent manner and no dark toxicity was observed. The cell death mechanisms triggered were evaluated by several assays and cell line-dependent results were found. Most SKBR-3 cells died by either necrosis or apoptosis. By contrast, in MCF-10A cells, necrotic cells and another cell population with characteristics of both necrosis and apoptosis were predominant. In this latter population, cell death was PARP-dependent and translocation of AIF to the nucleus was observed in some cells. These characteristics are related with parthanatos, being the first evidence of this type of regulated necrosis in the field of photodynamic therapy.

Abstract:Zn-containing porphyrins are intensely investigated for their ability to form reactive oxygen species and thereby being potent photosensitizers for use in photodynamic therapy (PDT). Some of the drawbacks of the PDT approach, such as unspecific distribution, could be addressed by means of photosensitizer drug delivery systems. In this work, we synthesize and characterize new water-soluble gold nanoparticles (GNP) stabilized by a mixture of a polyethyleneglycol-containing thiol (to improve water solubility) and a new amphiphilic gemini-type pyridinium salt, which also acts as promotor of the incorporation of the anionic photosensitizer Na-ZnTCPP into the GNP.The obtained GNP have sizes between 7-10 nm, as observed by Transmission Electron Microscopy. The incorporation of the photosensitizer caused an increase in the hydrodynamic size, detected by Dynamic Light Scattering, as well as a shift in the Surface Plasmon Resonance peak on the GNP UV-visible absorption spectra. The presence of the photosensitizer in the GNP was corroborated using Fluorescence Spectroscopy. The amount of Na-ZnTCPP was found to be 327 molecules per GNP. The porphyrincontaining Na-ZnTCPP-1·GNP showed good enhanced ability to produce singlet oxygen, compared to free Na-ZnTCPP. Their cytotoxicity and phototoxicity were investigated in vitro using two different human breast cell lines, one of tumoral origin (SKBR-3) and another of normal epithelium origin (MCF-10A). SKBR-3 cells showed higher sensitivity to Na-ZnTCCP and Na-ZnTCPP-1·GNP in dark conditions. After irradiation, no significant differences were observed between both cell lines except for 1µM Na-ZnTCCP-1·GNP where SKBR-3 cells were also more sensitive.

Photodynamic therapy (PDT) is a cancer treatment modality based on the administration of a photosensitizer (PS), which accumulates preferentially in tumor cells. Subsequent irradiation of the neoplastic area triggers a cascade of photochemical reactions that leads to the formation of highly reactive oxygen species responsible for cell inactivation. Photodynamic treatments in vitro are performed with the PS, zinc-phthalocyanine (ZnPc). The PS is near the plasma membrane during uptake and internalization. Inactivation clearly occurs by a necrotic process, manifested by nuclear pyknosis, negative TUNEL and Annexin V assays and non-relocation of cytochrome c. In contrast, by increasing the incubation time, ZnPc is accumulated in the Golgi apparatus and produces cell inactivation with characteristics of apoptosis and necrosis: TUNEL positive, relocated cytochrome c and negative Annexin V assay. This type of death produces a still undescribed granulated nuclear morphology, which is different from that of necrosis or apoptosis. This morphology is inhibited by necrostatin-1, a specific inhibitor of regulated necrosis.