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 type-dependent 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.

Background: Electrochemotherapy and gene electrotransfer are novel promising treatments employing locally applied high electric pulses to introduce chemotherapeutic drugs into tumor cells or genes into target cells based on the cell membrane electroporation. The main focus of this paper was to calculate analytically and numerically local electric field distribution inside the treated tissue in two dimensional (2D) models for different plate and needle electrode configurations and to compare the local electric field distribution to parameter U/d, which is widely used in electrochemotherapy and gene electrotransfer studies. We demonstrate the importance of evaluating the local electric field distribution in electrochemotherapy and gene electrotransfer. Methods: We analytically and numerically analyze electric field distribution based on 2D models for electrodes and electrode configurations which are most widely used in electrochemotherapy and gene electrotransfer. Analytical calculations were performed by solving the Laplace equation and numerical calculations by means of finite element method in two dimensions. Results: We determine the minimal and maximal E inside the target tissue as well as the maximal E over the entire treated tissue for the given electrode configurations. By comparing the local electric field distribution calculated for different electrode configurations to the ratio U/d, we show that the parameter U/d can differ significantly from the actual calculated values of the local electric field inside the treated tissue. By calculating the needed voltage to obtain E > U/d inside the target tissue, we showed that better electric field distribution can be obtained by increasing the number and changing the arrangement of the electrodes. Conclusion: Based on our analytical and numerical models of the local electric field distribution we show that the applied voltage, configuration of the electrodes and electrode position need to be chosen specifically for each individual case, and that numerical modeling can be used to optimize the appropriate electrode configuration and adequate voltage. Using numerical models we further calculate the needed voltage for a specific electrode configuration to achieve adequate E inside the target tissue while minimizing damages of the surrounding tissue. We present also analytical solutions, which provide a convenient, rapid, but approximate method for a pre-analysis of electric field distribution in treated tissue.

Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (N_DNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.

Triple naegative breast cancer has an increased rate of distant metastasis and consequently poor prognosis. To metastasize, breast cancer cells must detach from the main tumour mass and resist anoikis, a programmed cell death induced by lack of cell-extracellular matrix communication. Although cancer cells must detach to metastasize in vivo, the viability of floating cancer cells in vitro is rarely investigated. Here we show that co-treatment of anoikis-resistant MDA-MB-231 cells with metformin and 2-deoxy-D-glucose (2-DG) increased the percentage of floating cells, of which about 95% were viable. Floating cells resumed their proliferation once they were reseeded in the pharmacological compound-free medium. Similar effects on detachment were observed on anoikis-prone MCF-7 cells. Co-treatment of MDA-MB-231 cells with metformin and 2-DG induced a strong activation of AMP-activated protein kinase (AMPK), which was reduced by AMPK inhibitor compound C that prevented detachment of MDA-MB-231 cells. However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells, indicating that AMPK activation is necessary but not sufficient for triggering detachment of cancer cells. Our results demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents.