Jasna Lojk

Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate.

In recent years, nanoparticles (NPs) and related applications have become an intensive area of research, especially in the biotechnological and biomedical fields, with magnetic NPs being one of the promising tools for tumor treatment and as MRI-contrast enhancers. Several internalization and cytotoxicity studies have been performed, but there are still many unanswered questions concerning NP interactions with cells and NP stability. In this study, we prepared functionalized magnetic NPs coated with polyacrylic acid, which were stable in physiological conditions and which were also nontoxic short-term. Using fluorescence, scanning, and transmission electron microscopy, we were able to observe and determine the internalization pathways of polyacrylic acid–coated NPs in Chinese hamster ovary cells. With scanning electron microscopy we captured what might be the first step of NPs internalization – an endocytic vesicle in the process of formation enclosing NPs bound to the membrane. With fluorescence microscopy we observed that NP aggregates were rapidly internalized, in a time-dependent manner, via macropinocytosis and clathrin-mediated endocytosis. Inside the cytoplasm, aggregated NPs were found enclosed in acidified vesicles accumulated in the perinuclear region 1 hour after exposure, where they stayed for up to 24 hours. High intracellular loading of NPs in the Chinese hamster ovary cells was obtained after 24 hours, with no observable toxic effects. Thus polyacrylic acid–coated NPs have potential for use in biotechnological and biomedical applications.

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

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.

siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.

Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.

Increased endocytosis of magnetic nanoparticles into cancerous urothelial cells versus normal urothelial cells

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 rhodamine B isothiocyanate-labelled 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 papillary and invasive urothelial neoplasm cells by ruffling of the plasma membrane and engulfment of NP aggregates by macropinocytotic mechanism. Transmission electron microscopy (TEM) and spectrophotometric analyses showed that the efficacy of NPs delivery into normal urothelial cells and intercellular space is largely restricted, while it is significantly higher in cancer urothelial cells. Moreover, we showed that the quantification of fluorescent NP internalization in cells or tissues based on fluorescence detection could be misleading and overestimated without TEM analysis. Our findings contribute to the understanding of endocytosis-mediated cellular uptake of NPs in cancer urothelial cells and reveal a highly selective mechanism to distinguish cancer and normal urothelial cells.

Cell stress response to two different types of polymer coated cobalt ferrite nanoparticles

Potential nanoparticle (NP) toxicity is one of crucial problems that limit the applicability of NPs. When designing NPs for biomedical and biotechnological applications it is thus important to understand the mechanisms of their toxicity. In this study, we analysed the stress responses of previously designed polyacrylic acid (PAA) and polyethylenimine (PEI) coated NPs on primary human myoblasts (MYO) and B16 mouse melanoma cell line. Negatively charged PAA did not induce cell toxicity, reactive oxygen species (ROS) or activate the transcription factor NF-κB in either cell line even at high concentrations (100μg/ml). On the other hand, positively charged PEI NPs induced a concentration dependent necrotic cell death and an increase in ROS following 24h incubation already at low concentrations (>4μg/ml). Moreover, PEI NPs induced NF-κB activation 15-30min after incubation in MYO cells, most probably through activation of TLR4 receptor. Interestingly, there was no NF-κB response to PEI NPs in B16 cells.

Comparison of two automatic cell-counting solutions for fluorescent microscopic images

Cell counting in microscopic images is one of the fundamental analysis tools in life sciences, but is usually tedious, time consuming and prone to human error. Several programs for automatic cell counting have been developed so far, but most of them demand additional training or data input from the user. Most of them do not allow the users to online monitor the counting results, either. Therefore, we designed two straightforward, simple‐to‐use cell‐counting programs that also allow users to correct the detection results. In this paper, we present the Cellcounter and Learn123 programs for automatic and semiautomatic counting of objects in fluorescent microscopic images (cells or cell nuclei) with a user‐friendly interface. Although Cellcounter is based on predefined and fine‐tuned set of filters optimized on sets of chosen experiments, Learn123 uses an evolutionary algorithm to determine the adapt filter parameters based on a learning set of images. Cellcounter also includes an extension for analysis of overlaying images. The efficiency of both programs was assessed on images of cells stained with different fluorescent dyes by comparing automatically obtained results with results that were manually annotated by an expert. With both programs, the correlation between automatic and manual counting was very high (R2 < 0.9), although Cellcounter had some difficulties processing images with no cells or weakly stained cells, where sometimes the background noise was recognized as an object of interest. Nevertheless, the differences between manual and automatic counting were small compared to variations between experimental repeats. Both programs significantly reduced the time required to process the acquired images from hours to minutes. The programs enable consistent, robust, fast and accurate detection of fluorescent objects and can therefore be applied to a range of different applications in different fields of life sciences where fluorescent labelling is used for quantification of various phenomena. Moreover, Cellcounter overlay extension also enables fast analysis of related images that would otherwise require image merging for accurate analysis, whereas Learn123s evolutionary algorithm can adapt counting parameters to specific sets of images of different experimental settings.

Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic nanoparticles in CHO cells

Polyethyleneimine (PEI) is a polycationic compound frequently used as a transfection agent. However, cytotoxicity of PEI and PEI-coated nanoparticles (PEI NPs) is still a major obstacle in its use. In this study we report a method for reducing cytotoxicity of PEI NPs by addition of glutathione in NPs synthesis. Glutathione reduced cytotoxic effects for at least 30% and decreased observed oxidative stress response compared to standard formulation. Results showed that the effect was partially due to reduced zeta potential and partially due to protective antioxidant properties of glutathione. Addition of glutathione to cell culture media with concurrent exposure to PEI NPs proved to be insufficient for cytotoxicity reduction. Additionally, we compared internalization pathways of both PEI NPs and GSH NPs. NPs were only found in endosomes and no NPs were found free in the cytosol, as would be expected according to so called proton sponge hypothesis.

Automatic Cell Counter for cell viability estimation

Despite several methods that exist in different fields of life sciences, certain biotechnological applications still require microscopic analysis of the samples and in many instances, counting of cells. Some of those are drug delivery, transfection or analysis of mechanism fluorescent probes are used to detect cell viability, efficiency of a specific drug delivery or some other effect. For analysis and quantification of these results it is necessary to either manually or automatically count and analyze microscope images. However, in everyday use many researchers still count cells manually since existing solutions require either some specific knowledge of computer vision and/or manual fine tuning of various parameters. Here we present a new software solution (named CellCounter) for automatic and semi-automatic cell counting of fluorescent microscopic images. This application is specifically designed for counting fluorescently stained cells. The program enables counting of cell nuclei or cell cytoplasm stained with different fluorescent stained. This simplifies image analysis for several biotechnological applications where fluorescent microscopy is used. We present results and validate the presented automatic cell counting program for cell viability application. We give empirical results showing the efficiency of the proposed solution by comparing manual counts with the results returned by automated counting. We also show how the results can be further improved by combining manual and automated counts.

Automatic adaptation of filter sequences for cell counting

Manual cell counting in microscopic images is usually tedious, time consuming and prone to human error. Several programs for automatic cell counting have been developed so far, but most of them demand some specific knowledge of image analysis and/or manual fine tuning of various parameters. Even if a set of filters is found and fine tuned to the specific application, small changes to the image attributes might make the automatic counter very unreliable. The goal of this article is to present a new application that overcomes this problem by learning the set of parameters for each application, thus making it more robust to changes in the input images. The users must provide only a small representative subset of images and their manual count, and the program offers a set of automatic counters learned from the given input. The user can check the counters and choose the most suitable one. The resulting application (which we call Learn123) is specifically tailored to the practitioners, i.e. even though the typical workflow is more complex, the application is easy to use for non-technical experts.