Water-soluble upconversion nanoparticles (UCNPs), based on polyvinylpyrrolidone (PVP)-coated NaYF:Er,Yb,Gd, with various concentrations of Gd ions and relatively high upconversion efficiencies, were synthesized. The internalization and cytotoxicity of the thus obtained UCNPs were evaluated in three cell lines (HeLa, HEK293 and astrocytes). No cytotoxicity was observed even at concentrations of UCNPs up to 50 μg ml. The fate of the UCNPs within the cells was studied by examining their upconversion emission spectra with confocal microscopy and confirming these observations with transmission electron microscopy. It was found that the cellular uptake of the UCNPs occurred primarily by clathrin-mediated endocytosis, whereas they were secreted from the cells via lysosomal exocytosis. The results of this study, focused on the mechanisms of the cellular uptake, localization and secretion of UCNPs, demonstrate, for the first time, the co-localization of UCNPs within discrete cell organelles.

Osteosarcoma (OSA) is the most common malignant bone neoplasia in humans and dogs. In dogs, treatment consists of surgery in combination with chemotherapy (mostly carboplatin and/or doxorubicin (Dox)). Chemotherapy is often rendered ineffective by multidrug resistance. Previous studies have revealed that Dox conjugated with 4 nm glutathione-stabilized gold nanoparticles (Au-GSH-Dox) enhanced the anti-tumor activity and cytotoxicity of Dox in Dox-resistant feline fibrosarcoma cell lines exhibiting high P-glycoprotein (P-gp) activity. The present study investigated the influence of Au-GSH-Dox on the canine OSA cell line D17 and its relationship with P-gp activity. A human Dox-sensitive OSA cell line, U2OS, served as the negative control. Au-GSH-Dox, compared to free Dox, presented a greater cytotoxic effect on D17 (IC50 values for Au-GSH-Dox and Dox were 7.9 μg/mL and 15.2 μg/mL, respectively) but not on the U2OS cell line. All concentrations of Au-GSH (ranging from 10 to 1000 μg/mL) were non-toxic in both cell lines. Inhibition of the D17 cell line with 100 μM verapamil resulted in an increase in free Dox but not in intracellular Au-GSH-Dox. The results indicate that Au-GSH-Dox may act as an effective drug in canine OSA by bypassing P-gp.

Due to the high toxicity of currently used chemotherapeutics, novel methods of cancer treatment are needed. Gold nanoparticles (AuNPs) seem to be an interesting alternative due to penetration through biological membranes and systemic barriers. AuNPs as carriers of chemotherapeutics allow for reduced concentrations whilst maintaining the expected effect, and thus reducing the costs of therapy and adverse effects. We synthesized AuNPs stabilized with reduced glutathione (GSH) and conjugated with doxorubicin (DOX), gemcitabine (GEM) or cytarabine (CTA). This is the first study in which cytarabine-AuNPs were synthesized and characterized. Transmission electron microscopy (TEM), thermogravimetric analysis (TGA), nuclear magnetic resonance spectroscopy (NMR) and highperformance liquid chromatography (HPLC) were used to chemically characterize obtained nanoparticles. Antitumor activity and safety of application were assessed by MTT assay in in vitro model (human osteosarcoma cells -143B, human osteoblast-hFOB1.19, breast cancer cells -MCF7, breast epithelial cells -MCF10A, pancreatic cancer cells -PANC-1, and pancreatic cells -hTERT-HPNE cells). We have shown that cellular response varies according to the type and concentration of AuNPs. At some concentrations, we were able to show selective cytotoxicity of our AuNPs conjugates only to cancer cell lines. Synthesized nanoparticles were more cytotoxic to tumor cell lines than chemotherapeutics alone.

Feline injection site sarcomas (FISS) are malignant skin tumors with high recurrence rates despite the primary treatment of radical surgical resections. Adjunctive radiotherapy or chemotherapy with doxorubicin is mostly ineffective. Cellular and molecular causes of multidrug resistance, specific physio-chemical properties of solid tumors impairing drug transport, and the tumor microenvironment have been indicated for causing standard chemotherapy failure. Gold nanoparticles are promising imaging tools, nanotherapeutics, and drug delivery systems (DDS) for chemotherapeutics, improving drug transport within solid tumors. This study was conducted to assess the distribution of 4-nm glutathione-stabilized gold nanoparticles in FISS and their influence on kidney and liver parameters in nude mice. The role of gold nanoparticles as a doxorubicin DDS in FISS was examined to determine the potential reasons for failure to translate results from in vitro to in vivo studies. Grade III tumors characterized by a large area of necrosis at their core displayed positive immuneexpression of tumor-associated macrophages (TAM) at both the periphery and within the tumor core near the area of necrosis. Gold nanoparticles did not cause necrosis at the injection site and had no negative effect on liver and kidney parameters in nude mice. Gold nanoparticles accumulated in the tumor core and at the periphery and co-internalized with TAM—an important observation and potential therapeutic target warranting further investigation. The large area of necrosis and high immunoexpression of TAM, indicating “pro-tumor macrophages”, may be responsible for FISS tumor progression and therapeutic failure. However, further studies are required to test this hypothesis.

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