Alteration of zeta potential and cell viability in rat-derived L6 skeletal muscle cells and H9c2 cardiomyocytes: A study with submicron polystyrene particles.

BACKGROUND: Microand nanoplastics pollution can cause substantial damage to ecosystems. Since scientists have focused mainly on their impact on aquatic environments, less attention has been paid to the accumulation of polymer particles in terrestrial organisms. OBJECTIVES: We checked if submicron (<5 mm) polystyrene (PS) particles, which can accumulate in living organisms, lead to changes in the physicochemical properties of mammalian cell membranes. MATERIAL AND METHODS: The influence of submicron PS particles on the properties of rat-derived L6 myocytes and H9c2 cardiomyocytes was analyzed. Non-functionalized and amine-functionalized PS particles of 100 nm and 200 nm in diameter were used. The MTT assay was performed to evaluate the viability of the polymers-treated cells. The effect of short (6 h) and prolonged (48 h) incubation with different concentrations of PS particles on the cell's zeta (ζ) potential was examined with the electrophoretic light scattering technique (ELS). Polystyrene particles' physicochemical characteristics (size and stability) were performed using dynamic light scattering (DLS) and electrophoretic light scattering methods. RESULTS: The results show that submicron PS particles affect cell viability and cause changes in the physiochemical parameters of rat cell membranes. Differences were observed depending on the origin of the cells. We observed doseand time-dependent alterations in the studied parameters after submicron PS particle incubation in L6 myotubes and H9c2 cardiomyocytes. CONCLUSIONS: The size and modification of PS particle surfaces determine the extent to which they affect the analyzed properties of rat cardiomyocytes and myocytes membranes.

Exposure to polystyrene nanoparticles leads to changes in the zeta potential of bacterial cells.

Polymer molecules, the main components of plastics, are an emerging pollutants in various environmental compartments (water, air, soil) that may induce several ecotoxicological effects on live organisms. Therefore, understanding how plastic particles interact with bacterial cell membranes is crucial in analysing their associated risks in ecosystems and human microbiota. However, relatively little is known about the interaction between nanoplastics and bacteria. The present work focuses on Staphylococcus aureus and Klebsiella pneumoniae, representing the Gram-positive and Gram-negative bacteria respectively, exposed to 100 nm diameter polystyrene nanoparticles (PS NPs). The nanoparticles attach to the cells' membranes of both bacteria, changing their electrical charge, but without the effect of killing the cells. PS NPs caused a change in zeta potential values (both species of bacterial strains), dependent on particle concentration, pH, as well as on exposure time of bacteria to them. Through the application of AFM and FTIR techniques, the presence of PS NPs on bacterial surfaces was detected, suggesting the affinity of the particles to bacterial components, but without any changes in the morphology of the tested bacteria. The zeta potential can be more widely used in the study of interactions between nanostructures and cells.

The Effect of Submicron Polystyrene on the Electrokinetic Potential of Cell Membranes of Red Blood Cells and Platelets.

In recent years, many scientists have studied the effects of polymer micro- and nanostructures on living organisms. As it turns out, plastic can be a component of the blood of livestock, eaten by humans around the globe. Thus, it seems important to investigate possible changes in the physicochemical parameters and morphology of the cell membranes of blood morphotic elements (red blood cells and platelets) under the influence of polymer particles. The article presents research in which cell membranes were exposed to plain polystyrene (PS) and amino-functionalized polystyrene (PS-NH2) of two different sizes. The polymers were characterized by infrared spectroscopy and dynamic light-scattering methods. To analyze possible changes caused by polymer exposure in the structure of the membranes, their zeta potentials were measured using the electrophoretic light-scattering technique. The concentration of the polymers, as well as the exposure time, were also taken into the consideration during the research. Based on the obtained results, we concluded that 100 and 200 nm PS, as well as 100 nm PS-NH2, internalize into the cells. On the contrary, 200 nm PS-NH2 particles attach to cell membranes. Our study clearly shows that particle size and surface chemistry determine the interaction with biological membranes.

The influence of the pH on the incorporation of caffeic acid into biomimetic membranes and cancer cells.

Caffeic acid (CA) is a phenolic compound synthesized by all plant species. It constitutes the main hydroxycinnamic acid found in human diet and presents a variety of beneficial effects including anticancer activity. Current data suggests essential role of the interplay between anticancer drugs and the cell membrane. Given this, biophysical interactions between CA and cancer cells or biomimetic membranes were investigated. Glioblastoma cell line U118MG and colorectal adenocarcinoma cell line DLD-1, as well as lipid bilayers and liposomes, were used as in vitro models. Electrophoretic light scattering was used to assess the effect of CA on the surface charge of cancer cells and liposomal membranes. Electrochemical impedance spectroscopy was chosen to evaluate CA-dependent modulatory effect on the electrical capacitance and electrical resistance of the bilayers. Our results suggest that CA fulfills physicochemical criteria determining drug-like properties of chemical compounds, and may serve as a potential cytostatic agent in cancer treatment.

Experimental and Theoretical Approaches to Describing Interactions in Natural Cell Membranes Occurring as a Result of Fatal Alcohol Poisoning.

We propose herein a theoretical model describing the effect of fatal ethanol poisoning on the equilibria between cell membranes and the surrounding ions. Using this model, we determined the parameters characterizing the interaction between the electrolyte solution's ions and the functional groups on the blood cells' surface. Via the application of mathematical equations, we calculated the total surface concentrations of the acidic and basic groups, cA and cB, and their association constants with solution ions, KAH and KBOH. Using the determined parameters and mathematical equations' values, we calculated the theoretical surface charge density values. We verified the proposed model by comparing these values with experimental data, which were selected based on measurements of the electrophoretic mobility of erythrocyte and thrombocyte membranes. Compatibility of the experimental and theoretical surface charge density values was observed in the range of pH 2-8, while deviations were observed at higher pH values.

Effect of Selected Anionic and Cationic Drugs Affecting the Central Nervous System on Electrical Properties of Phosphatidylcholine Liposomes: Experiment and Theory.

Interactions between phospholipid membranes and selected drugs affecting the central nervous system (CNS) were investigated. Small, unilamellar liposomes were used as biomimetic cell membrane models. Microelectrophoretic experiments on two-component liposomes were performed using the electrophoretic light scattering technique (ELS). The effect of both positively (perphenazine, PF) and negatively (barbituric acid, BA) charged drugs on zwitterionic L-α-phosphatidylcholine (PC) membranes were analyzed. Experimental membrane surface charge density (δ) data were determined as a function of pH. Quantitative descriptions of the adsorption equilibria formed due to the binding of solution ions to analyzed two-component membranes are presented. Binding constants of the solution ions with perphenazine and barbituric acid-modified membranes were determined. The results of our research show that both charged drugs change surface charge density values of phosphatidylcholine membranes. It can be concluded that perphenazine and barbituric acid are located near the membrane surface, interacting electrostatically with phosphatidylcholine polar heads.

Electrophoretic Light Scattering and Electrochemical Impedance Spectroscopy Studies of Lipid Bilayers Modified by Cinnamic Acid and Its Hydroxyl Derivatives.

Pharmacological efficiency of active compounds is largely determined by their membrane permeability. Thus, identification of drug-membrane interactions seems to be a crucial element determining drug-like properties of chemical agents. Yet, knowledge of this issue is still lacking. Since chemoprevention based on natural compounds such as cinnamic acid (CinA), p-coumaric acid (p-CoA) and ferulic (FA) is becoming a strong trend in modern oncopharmacology, determination of physicochemical properties of these anticancer compounds is highly important. Here, electrophoretic light scattering and impedance spectroscopy were applied to study the effects of these phenolic acids on electrical properties of bilayers formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) or DOPC-PS mixture. After phenolic acid treatment, the negative charge of membranes increased in alkaline pH solutions, but not in acidic ones. The impedance data showed elevated values of both the electrical capacitance and the electrical resistance. We concluded that at acidic pH all tested compounds were able to solubilize into the membrane and permeate it. At neutral and alkaline pH, the CinA could be partially inserted into the bilayers, whereas p-CoA and FA could be anchored at the bilayer surface. Our results indicate that the electrochemical methods might be crucial for predicting pharmacological activity and bioavailability of phenolic acids.

Monitoring of the Surface Charge Density Changes of Human Glioblastoma Cell Membranes upon Cinnamic and Ferulic Acids Treatment.

Cinnamic acid (CA) and ferulic acid (FA) are naturally occurring phenolic acids claimed to exert beneficial effects against disorders related to oxidative stress, including cancer. One such malignancy that still remains a therapeutic challenge mainly due to its heterogeneity and inaccessibility to therapeutic agents is Glioblastoma multiforme (GBM). Here, the influence of CA and FA on the surface charge density of human GBM cell line LN-229 was studied using the electrophoretic light scattering technique. Also, the cytotoxicity of both phenolic acids was determined by metabolic activity-assessing tetrazolium test (MTT) analysis after exposure to CA and FA for 24 h and 48 h. Results showed that both compounds reduced cell viability of LN-229 cells, with more pronounced effect evoked by CA as reflected in IC50 values. Further analyses demonstrated that, after treatment with both phenolic acids, the negative charge of membranes decreased at high pH values and the positive charge of the membranes increased at low pH values compared to the data obtained for untreated cells. Afterward, a four-equilibrium model was applied to estimate the total surface concentrations of both acidic and basic functional groups and their association constants with solution ions in order to calculate theoretical values of membrane surface charge densities. Then, the theoretical data were compared to the experimental data in order to verify the mathematical model. As such, our results indicate that application of electrochemical methods to determine specific drug-membrane interactions might be crucial for predicting their pharmacological activity and bioavailability.

The Modulating Effect of p-Coumaric Acid on The Surface Charge Density of Human Glioblastoma Cell Membranes.

p-Coumaric acid (p-CoA), a phenolic acid belonging to the hydroxycinnamic acids family, is a compound with tentative anticancer potential. Microelectrophoretic mobility measurements conducted at various pH values of electrolyte solution were applied to study p-CoA effects on electrical properties of human glioblastoma cell membranes. The obtained results demonstrated that after the p-CoA treatment, the surface charge density of cancer cells changed in alkaline pH solutions, while no noticeable changes were observed in cell membranes incubated with p-CoA compared to control at acidic pH solutions. A four-equilibrium model was used to describe the phenomena occurring on the cell membrane surface. The total surface concentrations of both acidic and basic functional groups and their association constants with solution ions were calculated and used to define theoretical curves of membrane surface charge density versus pH. The resulting theoretical curves and the experimental data were compared to verify the reliability and validity of the adopted model. The deviation of both kinds of data obtained at a higher pH may be caused by disregarding interactions between the functional groups of cancer cells. Processes occurring in the cell membranes after their incubation with p-CoA can lead to disorders of existing equilibria, which result in changes in values of the parameters describing these equilibria.

The modulating effect of lipid bilayer/p-coumaric acid interactions on electrical properties of model lipid membranes and human glioblastoma cells.

Biological membranes are one of the most important elements of living cells determining their permeability to the active compounds. Still, little is known about the drug-membrane interactions in terms of pharmacological properties of potential drugs. Chemoprevention based on natural compounds is becoming a strong trend in modern oncopharmacology, and p-coumaric acid (p-CoA) is one such compound with tentative anticancer activity. The microelectrophoretic mobility measurements and electrochemical impedance spectroscopy were applied to study the effects of p-CoA on electrical properties of liposomes, spherical bilayers, and human glioblastoma cell membranes. Our results demonstrated that after treatment with p-CoA, the surface charge of LBC3, LN-229 and LN-18 cell lines was significantly changed in alkaline pH solutions, but not in acidic pH solutions. In contrast, no changes in surface charge density values were registered for phosphatidylethanolamine liposomal membranes and A172 cell membranes after p-CoA treatment. The impedance data showed an increase in values of both the electrical capacitance and the electrical resistance, indicating that p-CoA can be partially inserted into the phosphatidylcholine bilayers. The MTT assay showed cell line-dependent cytotoxic effect of p-CoA. Further molecular analyses revealed the ATP depletion and gene transcription modulation, which might indicate organelle membrane-crossing potential of p-CoA. These results suggest, that changes in surface charge of membranes of living cells not only might be potential predictor of membrane permeability, but also indicate differential composition of cell membranes in various cell lines. Thus further multidirectional analyses are required to implement electrochemical methods as standard testing procedures during drug development process.

Theoretical Considerations and the Microelectrophoresis Experiment on the Influence of Selected Chaotropic Anions on Phosphatidylcholine Membrane Surface Charge Density.

Influence of sodium salts of selected chaotropic anions from the Hofmeister series (NaCl, NaBr, NaNO3, NaI) on the surface charge density of phosphatidylcholine membranes was studied. Small unilamellar lipid vesicles were used as a model system in the investigations. The theoretical and experimental approach to the interactions between inorganic anions and phosphatidylcholine membranes is presented. Experimental membrane surface charge densities data were determined as a function of pH of the aqueous electrolytes using microelectrophoresis method. The quantitative description of the interactions between zwitterionic phosphatidylcholine membrane and monovalent anions is presented. The equilibria constants of the binding of solution ions onto phospholipid surface were calculated. Knowledge of these parameters was essential to determine the theoretical membrane surface charge density values. The theoretical data were compared to the experimental ones in order to verify the mathematical model. Both approaches indicate that the anion-phosphatidylcholine membrane interaction increases with the size of the anion. The adsorption of chaotropic anions to membranes was found to follow the Hofmeister series I- > NO3- > Br- > Cl-.

The effect of quercetin on the electrical properties of model lipid membranes and human glioblastoma cells.

Quercetin is a naturally-occurring flavonoid claimed to exert many beneficial health effects. In this report, the influence of quercetin on the surface charge of phosphatidylcholine liposomes and human glioblastoma LN-229 and LN-18 cells was studied using microelectrophoretic mobility measurements. The effect of quercetin on the electrical resistance and capacitance of bilayer lipid membranes was analyzed via electrochemical impedance spectroscopy. The results showed that after flavonoid treatment, the cell lines demonstrated changes in surface charge only in alkaline pH solutions, whereas there were no significant alterations in quercetin-treated vs. control cells in acidic pH solutions. The same tendency was found for liposomal membranes proving that quercetin insertion into membranes is strongly pH-dependent. Capacitance and resistance measurements conducted in acidic electrolyte solutions demonstrated an increase in both electrical parameters, indicating an increased amount of quercetin inserted into the bilayers. Moreover, the cytotoxic effect of quercetin confirms that the flavonoid enters the cells and perturbs the proliferation of LN-229 and LN-18 glioblastoma cell lines. As such, our results indicate that the specific localization of quercetin, membrane-bound or cell-entering, might be crucial for its pharmacological activity. However, further studies are necessary prior to applying these physicochemical measurements as standard methods of evaluating drug activity.

Binding of trivalent metal ions (Al3+, In3+, La3+) with phosphatidylcholine liposomal membranes investigated by microelectrophoresis.

Interactions between trivalent metal ions (Al3+, In3+, La3+) and phosphatidylcholine (PC) liposomes are studied by microelectrophoresis. The dependence of the PC membrane surface charge density and zeta potential on [Formula: see text] ([Formula: see text] range from 2 to 10) of the aqueous metal chloride solutions is determined. The obtained results indicate the adsorption of Al3+, In3+ and La3+ ions on phosphatidylcholine model membranes, leading to changes in the electrical properties of the membranes. The theoretical considerations on equilibria occurring between phosphatidylcholine liposomal membrane and trivalent metal ions are presented. A mathematical model describing the interactions in a quantitative way is proposed.

The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines.

Silica nanoparticles (SiNPs) are one of the most commonly used nanomaterials in various medical applications. However, possible mechanisms of the toxicity caused by SiNPs remain unclear. The study presented here provides novel information on molecular and cellular effects of SiNPs in glioblastoma LBC3 and LN-18 cells. It has been demonstrated that SiNPs of 7 nm, 5-15 nm and 10-20 nm induce time- and dose-dependent cytotoxicity in LBC3 and LN-18 cell lines. In contrast to glioblastoma cells, we observed only weak reduction in viability of normal skin fibroblasts treated with SiNPs. Furthermore, in LBC3 cells treated with 5-15 nm SiNPs we noticed induction of apoptosis and necrosis, while in LN-18 cells only necrosis. The 5-15 nm SiNPs were also found to cause oxidative stress, a loss in mitochondrial membrane potential, and changes in the ultrastructure of the mitochondria in LBC3 cells. Quantitative real-time PCR results showed that in LBC3 cells the mRNA levels of pro-apoptotic genes Bim, Bax, Puma, and Noxa were significantly upregulated. An increase in activity of caspase-9 in these cells was also observed. Moreover, the activation of SiNP-induced autophagy was demonstrated in LBC3 cells as shown by an increase in LC3-II/LC3-I ratio, the upregulation of Atg5 gene and an increase in AVOs-positive cells. In conclusion, this research provides novel information concerning molecular mechanisms of apoptosis and autophagy in LBC3 cells.

Association of alkali metal cations with phosphatidylcholine liposomal membrane surface.

Interactions of alkali metal cations (Li+, Na+, K+, Cs+) with phosphatidylcholine (PC) liposomal membranes were investigated through experimental studies and theoretical considerations. Using a microelectrophoresis technique, charge densities of experimental membrane surfaces were measured as a function of the pH of electrolyte solutions. Equilibria between the PC liposomal membranes and monovalent ions were mathematically analyzed and described quantitatively through a previously proposed theoretical model. Association constants between functional groups of PC and the studied ions were determined and used to define theoretical curves of membrane surface charge density versus pH. Theoretical and experimental data were compared to verify the model. The PC membrane was found to have the highest affinity for lithium ions, among the ions tested.

Equilibria Between Cell Membranes and Electrolyte Solution: Effect of Fatal Accidental Hypothermia.

Equilibria between the membranes of erythrocytes as well as thrombocytes and solution ions in fatal accidental hypothermia were analyzed using a theoretical four-equilibria model. The model was developed to determinate parameters characterizing cell membrane-surrounding ion interactions: the total surface concentrations of both acidic and basic groups C A, C B, and association constants K AH, K BOH. Knowledge of these parameters was necessary to calculate the theoretical values of surface charge density. The model was validated by curve-fitting the experimental data points to simulated data generated by the model. The experimental and theoretical surface charge density values agree at pH 2-8, at higher pH the deviation was observed.

Changes in Surface Charge Density of Blood Cells in Fatal Accidental Hypothermia.

The objective of this research was to evaluate postmortem changes concerning electric charge of human erythrocytes and thrombocytes in fatal accidental hypothermia. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells conducted at various pH values of electrolyte solution. The surface charge of erythrocyte membranes after fatal accidental hypothermia increased compared to the control group within whole range of experimental pH values. Moreover, a slight shift of the isoelectric point of erythrocyte membranes towards high pH values was observed. The surface charge of thrombocyte membranes in fatal accidental hypothermia decreased at low pH compared to the control group. However, at pH range 4-9, the values increased compared to the control group. The isoelectric point of thrombocyte membranes after fatal accidental hypothermia was slightly shifted towards low pH values compared to the control group. The observed changes are probably connected with the partial destruction and functional changes of the blood cell structure.

The effect of fatal carbon monoxide poisoning on the equilibria between cell membranes and the electrolyte solution.

The effect of fatal carbon monoxide poisoning on equilibria between cell membranes and surrounding ions was described using a theoretical four-equilibria model. The model was developed to obtain parameters characterizing the interactions between solution ions and erythrocyte or thrombocyte membrane surface. The parameters are the total surface concentrations of both acidic and basic groups C A, C B and their association constants with solution ions K AH, K BOH. These parameters were used to calculate the theoretical values of surface charge density. The model was validated by comparison of these values to experimental data, which were determined from the electrophoretic mobility measurements of the blood cells. The experimental and theoretical surface charge density values agree at pH 2-8, and at higher pH, the deviation was observed.

Microelectrophoretic investigation of the interactions between liposomal membranes formed from a phosphatidylcholine-phosphatidylglycerol mixture and monovalent ions.

In this paper, we characterized the interactions between two-component liposomal membranes and monovalent electrolyte ions. Liposomes were formed from neutral (phosphatidylcholine) and anionic (phosphatidylglycerol) lipids mixed in various ratios. Microelectrophoresis was used to determine the dependence of the membrane surface charge density on the p H of the electrolyte solution. Changes in the membrane electric charge caused by the adsorption of Na(+), Cl(-), H(+), and OH(-) ions were observed, and the equilibria among these ions and the phosphatidylcholine-phosphatidylglycerol membrane surface were quantified. We proposed a mathematical model for characterizing these equilibria. Using this model, together with experimental data of the membrane surface charge density, we determined association constants characterizing the equilibria. Knowledge of these parameters was necessary to calculate the theoretical curves of the model. We validated the model by curve-fitting the experimental data points to simulated data generated by the model.