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. Related publication: In vitro assessment of potential bladder papillary neoplasm treatment with functionalized polyethyleneimine coated magnetic nanoparticles, Acta Chimica Slovenica 64: 543-548, 2017
COBISS.SI-ID: 11794772
Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours' exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours' exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP-cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. International patent application was filed, our magnetic NPs were also used for endosomal separation in the paper published in Pohar J et al., Nature Communications 8, 15363 (2017). Best poster awards on 7th Conference on Experimental and Translational Oncology (CETO 2013)
COBISS.SI-ID: 31824089
Increased environmental pollution has been suggested as one of the possible causes for increased incidence of neurodegenerative and developmental disorders. 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 analyzed the influence of four types of NP (biomedical polyethylenimine coated cobalt ferrite (CoFe2O4) NPs (PEI) and industrial TiO2 P25, TiO2 N and SiO2 NPs), on intracellular localization and solubility of fused in sarcoma (FUS) and TAR-DNA binding protein 43 (TDP-43), that are important pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The results showed that the selected industrial TiO2 NPs induced a decrease in nuclear and increase in cytoplasmic TDP-43 and FUS concentrations, which is the expected cell’s reaction to stress, a small decrease in cell viability, but no ROS. Industrial TiO2 P25 NPs only induced a small increase in ROS. Industrial SiO2 NPs induced no changes on the cells, most probably due to high NP aggregation and consequently low internalization. PEI NPs, on the other hand, induced severe membrane damage and several unexpected changes: decrease in ROS, increase in nuclear FUS concentration and decrease in urea soluble fraction of FUS protein, which are the opposite to the expected stress induced changes, suggesting the responses might be connected through a common mechanism. 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 NP exposure on ALS and FTLD.
COBISS.SI-ID: 11728980
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 looked into the 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 activation of NF-kB transcription factor. PEI NPs, on the other hand, induce severe necrotic cell death through membrane damage, induce ROS formation and activation of NF-kB signaling 15-30 min after incubation. Experiments with inhibitors showed, that the activation of NF-kB was mediated through TLR4 receptor. In another study (COBISS ID 10990164), we tried to reduce the observed PEI NP toxicity by additional binding of glutathione (GSH) on the surface of PEI NPs. With the additional GSH coating, we wanted to 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 and transfection with GFP followed by fluorescence microscopy showed equal efficiency at higher viability using GSH formulation.
COBISS.SI-ID: 11689556
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 demonstrated the effect of the dispersion media on 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, α-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. 5 pure citations in first year.
COBISS.SI-ID: 11662676