| Sažetak | CILJ ISTRAŽIVANJA. Srebrne nanočestice (nanoAg) su jedne od najistraživanijih metalnih nanočestica. Zbog svoje biocidne aktivnosti sve se učestalije primjenjuju u dijagnostičke i terapijske svrhe, što zahtijeva ispitivanje njihove biokompatibilnosti. Cilj ovog istraživanja bio je ispitati biološke učinke različito funkcionaliziranih nanočestica srebra na stanice humanog imunološkog sustava, te standardizirati metode za procjenu imunokompatibilnosti u svrhu dizajna sigurnih nanomedicinskih proizvoda koji sadrže srebro. NACRT STUDIJE. Ova studija je presječna in vitro studija. Sve analize provedene su na izoliranim humanim mononuklearnim stanicama periferne krvi (hPBMC) koje predstavljaju reprezentativni primjerak krvnih imunoloških stanica. Metode za procjenu imunokompatibilnosti odabrane su prema ISO smjernicama za ispitivanje hemokompatibilnosti medicinskih uređaja, te su prilagođene ispitivanju učinaka nanomaterijala na humane imunološke stanice. ISPITANICI I METODE. Cjelokupno istraživanje provedeno je na limfocitima i monocitima izoliranim iz svježih uzoraka pune krvi dobivenih od ukupno 30 zdravih ispitanika. U svrhu istraživanja pripremljene su četiri vrste nanoAg-a sferičnog oblika promjera oko 10 nm, stabilizirane različitim površinskim omotačima: neutralnim (PVP), pozitivnim (PLL), negativnim (AOT) i proteinskim (BSA). Metodom protočne citometrije analizirani su učinci citotoksičnosti (internalizacija nano-Ag-a, indukcija apoptoze i nekroze, te oksidativnog stresa) i imunomodulacije. Za vizualni prikaz formacije nanoAg-a u nanostrukturu, unutarstanične lokalizacije, te citotoksičnosti nanoAg-a, korištene su različite vrste mikroskopije (TEM, CLSM, svjetlosna mikroskopija). Za evaluaciju genotoksičnosti korišten je alkalni komet test. REZULTATI. Sve testirane vrste nanoAg-a promijenile su morfologiju hPBMC stanica, inducirajući apoptozu i nekrozu proporcionalno koncentraciji i vremenu izloženosti. Najvišu toksičnost pokazale su pozitivno nabijene (PLL) i proteinske (BSA) nanoAg. Ulazak nanoAg-a u stanice rastao je proporcionalno primjenjenoj koncentraciji, te je bio najviši kod PLL-nanoAg-a. Sve vrste nanoAg-a u stanici uzrokovale su oksidativni stres produkcijom ROS-ova i oštećenjem mitohondrijske membrane. Ovisno o koncentraciji, sve vrste nanoAg-a su uzrokovale genotoksičnost. BSA- i PLL-nanoAg uzrokovali su aktivaciju B-limfocita, NK-stanica i monocita, T-limfociti se nisu značajno aktivirali, ali se smanjio broj virusnog CD4-receptora na površini pomagačkih T-limfocita. ZAKLJUČCI. Ova studija predstavlja sistematičnu i sveobuhvatnu analizu koja pokazuje kako različito funkcionalizirane nanočestice srebra mogu utjecati na humani imunološki sustav. Rezultati ove studije predstavljaju značajan doprinos procjeni sigurnosti medicinskih nano-proizvoda na bazi srebra. |
| Sažetak (engleski) | OBJECTIVES. Silver nanoparticles (AgNPs) are one of the most investigated metal-based nanomaterials. Their biocidal activity has boosted their application in both diagnostic and therapeutic medical procedures. Therefore, it is crucial to create biocompatible AgNPs. This study has tried to deepen scientific knowledge about biomedical safety of AgNPs, depending on their different surface properties, which affect human immune system. Also, this study has as its goal to standardize immunocompatiblity methods, in order to evaluate human-related safety of AgNPs. STUDY DESIGN. This study is designed as cross-sectional in vitro study. All procedures and analysis were performed in vitro, on human peripheral blood mononuclear cells (hPBMCs), as main representatives of circulating immunological cells. Immunocompatibility evaluation methods were applied according to ISO guidelines for biological evaluation of medical products and got adjusted, in order to evaluate medically-applied nanomaterials on human immune cells. MATERIALS AND METHODS. Mononuclear cells (lymphocytes and monocytes) were isolated from freshly drawn, whole blood samples of 30 healthy donors. For the study purposes, there were four, spherically shaped nanoAg, with 10nm diameter, prepared and stabilized with different coating: AgNPs encompassing protein (BSA), neutral (PVP), positive (PLL) and negative (AOT) agents, used for their surface stabilization. Cytotoxicity effects, including nanoparticle cellular uptake potential, apoptotic and necrotic cell death, induction of oxidative stress, damages of mitochondrial membrane and cell activation, were analyzed using flow cytometry method. Nanoformation, intracellular localization and cytotoxicity effects of AgNPs were visually confirmed by different types of microscopic methods (TEM, CLSM, light). Genotoxicity evaluation was performed by alkaline comet assay. Coating-, dose- and time-dependent cellular toxicity of AgNPs was assessed. RESULTS. All tested AgNPs altered morphology of freshly isolated hPBMCs, inducing apoptosis and necrosis in a dose- and time-dependent manner. The highest toxicity had positively-charged and protein-coated AgNPs. Cellular uptake of AgNPs was also dose-dependently increased and the highest was for positively charged AgNPs. All types of AgNPs caused the production of reactive oxygen species (ROS) and damaged mitochondrial membrane intracellularly. Depending on the dose, all AgNPs exhibited genotoxic potential. BSA- and PLL-AgNPs stimulated B-cells, as well as NK-cells and monocyte activation; there was no significant T-cell activation, but there was a depletion of virus CD4-receptor on T-helper cell surface. CONCLUSIONS. This study provides systematic and comprehensive data, showing how differently functionalized AgNPs may affect the human immune system. These results present a valuable scientific contribution to safety assessment of nanosilver-based, blood-contacting medical products. |
