Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored - the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs typedependent specificity of the protein corona. But more importantly, we are first to demonstrate that the dispersion media determines the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, ?-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs. The established protocols were a basis for all further studies where we analysed protein corona composition in human sear nad cell media in relation to the immune response to NPs observed on macrophages and PBMC cells. 17 citations (without autocitations) in two years
COBISS.SI-ID: 11662676
Numerous different types of nanoparticles (NPs) are being designed with properties suitable for various NP applications, among which magnetic nanoparticles are especially problematic due to their nondegradable core, which is not stable in physiological conditions. To overcome this problem, different coatings have been proposed, depending on the desired biomedical or biotechnological application, each with its unique advantages and disadvantages, which can affect the efficacy of NPs for certain applications. Thorough understanding of toxicity mechanisms is thus essential for further design and improvement of NP formulations. In this study, we analysed mechanisms of toxicity of two types of cobalt ferrite NPs, coated with either negatively charged polyacrylic acid (PAA) or positively charged polyethylenimine (PEI). Experiments on B16 cell line and primary human myoblasts showed that PAA coating does not affect cell viability, ROS or trigger the immune response through the activation of NF-kB transcription factor (central transcription factor of the immune response). PEI NPs, on the other hand, inducde severe necrotic cell death through membrane damage, induce ROS formation and activation of NF-kB signaling 15-30 min after incubation. Experiments with specific inhibitors showed, that the activation of NF-kB was mediated through TLR4 receptor. This is important in order to understand potential immune effects of nanoaprticles coated with specific polymers. In paralel study (COBISS ID 10990164), we developed and tested further functionalisation in order to reduce the observed PEI NP toxicity by additional binding of antioxidant glutathione (GSH) on the surface of PEI NPs. With the additional GSH coating, we partially reduce the surface charge of NPs and reduce ROS by GSH’s antioxidant activity. The experiments performed on Chinese hamster ovary cells (CHO) showed that the additional coating indeed reduced cell toxicity and ROS induction as well as reduced the depletion of cell’s total intracellular glutathione. TEM micrographs showed that internalization was achieved with or without modification. Transfection using NPs as carriers for pE GFP followed by fluorescence microscopy showed equal efficiency at higher viability using GSH formulation. Thus GSH coating can be used in in order to decrease negative effects on cell viability due to PEI toxcity. Releated publications: STROJAN, Klemen, LOJK, Jasna, BREGAR, Vladimir Boštjan, VERANIČ, Peter, PAVLIN, Mojca. Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic nanoparticles in CHO cells. Toxicology in vitro, Jun. 2017, vol. 41, str. 12-20, [COBISS.SI-ID 11689556]
COBISS.SI-ID: 11691348
The blood–urine barrier is the tightest and most impermeable barrier in the body and as such represents a problem for intravesical drug delivery applications. Differentiation-dependent low endocytotic rate of urothelial cells has already been noted; however, the differences in endocytosis of normal and cancer urothelial cells have not been exploited yet. Here we analysed the endocytosis of polyacrylic acid-coated cobalt ferrite nanoparticles (NPs) in biomimetic urothelial in vitro models, i.e., in highly and partially differentiated normal urothelial cells, and in cancer cells of the papillary and invasive urothelial neoplasm. We demonstrated that NPs enter only papillary and invasive urothelial neoplasm cells by macropinocytotic mechanism, while almost no NPs were found inside normal cells as observed by transmission electron microscopy (TEM). Our findings reveal a highly selective mechanism to distinguish cancer and normal urothelial cells and opens a posibility for selective targeting. On the basis of this study and analysis of protein corona composition we are testing new dispersion media for optimal uptake into cancer cells. Related papers: STROJAN, Klemen, LOJK, Jasna, BREGAR, Vladimir Boštjan, ERDANI-KREFT, Mateja, SVETE, Jurij, VERANIČ, Peter, PAVLIN, Mojca. In vitro assessment of potential bladder papillary neoplasm treatment with functionalized polyethyleneimine coated magnetic nanoparticles. Acta chimica slovenica, 2017, vol. 64, , 543-548, http://10.0.67.192/acsi.2016.2876, doi: 10.17344/acsi.2016.2876.[COBISS.SI-ID 11738452]
COBISS.SI-ID: 11794772
Increased environmental pollution has been suggested as one of the possible causes for increased incidence of neurodegenerative and developmental disorders. Through the environmental pollution, everyday consumer products and nanomedical applications, we are also exposed to various nanoparticles (NPs). Specific types of NPs have been shown to be able to cause neural damage in vivo through processes such as disruption of the blood-brain barrier, induction of neuroinflammation, increase in oxidative stress and protein aggregation. In this study, we analysed the influence of PEI-coated magnetic NPs designed for biotechnological applications and industrial SiO2, TiO2 N and TiO2 P25 NPs on intracellular localization and solubility of fused in farcoma (FUS) and TAR-DNA binding protein 43 (TDP-43) that are important pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). SH-SY5Y neuroblastoma cells and B16 mouse melanoma cells were exposed to NPs for 24 h and analysed using confocal microscopy and Western blot. Exposure to 50 µg/ml TiO2 N and 4 µg/ml PEI NPs in SH-SY5Y cells caused cell toxicity-induced changes in expression in different biochemical/cellular fractions for both FUS and TDP-43 proteins. TiO2 N induced a drop in nuclear levels of TDP-43 and increase in cytoplasmic levels of FUS, while PEI NPs increased nuclear levels of FUS. Furthermore, TiO2 N and PEI induced a reduction of FUS and TDP-43 quantity in the less soluble urea fraction. No formation of stress granules was observed. These results demonstrate that TiO2 N and PEI NPs can affect the behaviour of FUS and TDP-43 proteins; however, the changes were relatively minor compared to pathological changes even for the high NP concentrations (50 µg/ml) used in this study. The study shows that the selected NPs do not induce changes of FUS or TDP-43 and stress response in short-term in vitro conditions, however long term and accumulation studies are required to determine the long-term impact of NPs exposure on ALS and FTLD. The results were also dissiminated through the COST action BIONECA Mojca Pavlin MC, STMS - Jasna Lojk visit into Biotalentum, Budapest.
COBISS.SI-ID: 11728980
Due to the great variability of NPs formulations there is still no clear connection between physico-chemical properties (PCP), protein corona composition and nanotoxicity and/or immunogenicity of NPs We focused on the dependence between three aspects: PCP, protein corona composition and toxicological endpoints by vitro analysis on THP-1 macrophages. Eight types of engineered NPs relevant to everyday exposure were analyzed: SiO2 (Nanotesla Institute Ljubljana) used in cleaning materials, silver Ag (biopure nanoComposix), TiO2 21 nm (Sigma), ANATAZ TiO2, TiO2 NPs used in cosmetics: Dr. Adorable (DrA) TiO2, Naturally Balmy (N.B.) TiO2, and two of most commonly used TiO2 NPs: Food-grade (FG) TiO2 (Cake Stuff) and TiO2 P25. Polyacrylic acid (PAA) coated Cobalt-ferrite NPs were used as an example of biomedical NPs. For characterization of PCP hydrodynamic radius, Z-average and zeta potential were measured in water and relevant cell culture media. NPs impurities were determined by ICP-MS. Protein corona composition was determined by mass spectroscopy and analyzed as relative protein abundance and total proteins counts. Nanotoxicity and immunotoxicity was assessed trough different endpoints: viability, reactive oxygen species (ROS) and cytokine secretion (TNF?, IL-6, IL-8, IL-1ß). Dependence between measured parameters was analyzed by calculating Spearman’s correlation coefficient. We show that except of Ag NPs all tested TiO2 types and SiO2 exhibited relatively low toxicity and some immune response in terms of IL8 and Il-1b cytokines secretion at moderate NPs concentrations. The cytokines secretion was independent of the protein corona composition but correlated with the total protein count and size of the NPs in the culture media. The response of THP-1 macrophages suggests at least two mechanisms of observed immune response. Paper in preparation: Nanotoxicity and immunogenicity of engineered nanoparticles dependence on the physico-chemical properties and protein corona composition, avtorji: K. Strojan, J. Lojk, I. Hafner Bratkovič, A. Leonardi, I. Križaj, N. Drnovšek, S. Novak Krmpotič, P. Veranič, V.B. Bregar, M. Pavlin*, M. Pavlin oral talk on conference Bionanomed 2019.
COBISS.SI-ID: 11948372