Generation Dependent Effects and Entrance to Mitochondria of Hybrid Dendrimers on Normal and Cancer Neuronal Cells In Vitro.

Dendrimers as drug carriers can be utilized for drugs and siRNA delivery in central nervous system (CNS) disorders, including various types of cancers, such as neuroblastomas and gliomas. They have also been considered as drugs per se, for example as anti-Alzheimer's disease (AD), anti-cancer, anti-prion or anti-inflammatory agents. Since the influence of carbosilane-viologen-phosphorus dendrimers (SMT1 and SMT2) on the basic cellular processes of nerve cells had not been investigated, we examined the impact of two generations of these hybrid macromolecules on two murine cell lines-cancer cell line N2a (mouse neuroblastoma) and normal immortalized cell line mHippoE-18 (embryonic mouse hippocampal cell line). We examined alterations in cellular responses including the activity of mitochondrial dehydrogenases, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, and morphological modifications and fractions of apoptotic and dead cells. Our results show that both dendrimers at low concentrations affected the cancer cell line more than the normal one. Also, generation-dependent effects were found: the highest generation induced greater cytotoxic effects and morphological modifications. The most promising is that the changes in mitochondrial membrane potential and transmission electron microscopy (TEM) images indicate that dendrimer SMT1 can reach mitochondria. Thus, SMT1 and SMT2 seem to have potential as nanocarriers to mitochondria or anti-cancer drugs per se in CNS disorders.

Cytotoxic activity of genistein-8-C-glucoside form Lupinus luteus L. and genistein against human SK-OV-3 ovarian carcinoma cell line.

Genistein belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. Numerous in vitro studies suggest that isoflavones, particularly genistein, have both chemopreventive and chemotherapeutic potential in multiple tumor types. However, the molecular and cellular mechanisms of genistein effects on human ovarian cancer cells are still little known. In the present study, we investigated anticancer activity of genistein and its natural glucoside, genistein-8-C-glucoside isolated from flowers of Lupinus luteus L. We examined the effects of the two isoflavones alone or in combination on cultured human SK-OV-3 ovarian carcinoma cells. The cells were exposed to genistein and genistein-8-C-glucoside at various concentrations (1-90 µM) for 24 and 48 h. The cytotoxic and apoptotic properties of compounds were studied by the colorimetric 3-[4,5-2-yl]-2-5-diphenyltetrazolium bromide assay and the acridine orange/ethidium bromide staining technique. The morphological features of SK-OV-3 cells were examined by Nomarski differential interference contrast combined with a confocal laser scanning microscope. The level of ROS was evaluated with fluorescence probes: dichlorofluorescein-diacetate by flow cytometry. Changes in mitochondrial membrane potential were determined using 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide. Genistein-treatment and genistein-8-C-glucoside-treatment resulted in the inhibition of cell proliferation, induction of apoptotic cell death and loss of mitochondrial membrane potential. The present data provide the first evidence in vitro that genistein-8-C-glucoside and combination genistein-genistein-8-C-glucoside could be a potential chemotherapeutic candidate for ovarian cancer therapy.

Carbosilane dendrimers inhibit α-synuclein fibrillation and prevent cells from rotenone-induced damage.

This study investigates the role of carbosilane dendrimers in fibrillation of α-synuclein and prevention of the mouse hippocampal cell (mHippoE-18) from rotenone-induced damage. Examining the interaction between carbosilane dendrimers and α-synuclein, we found that the dendrimers inhibit fibril formation. We also investigated cell viability, the production of reactive oxygen species (ROS), and mitochondrial membrane potential. mHippoE-18 cells were preincubated with carbosilane dendrimers before rotenone was added. All the dendrimers possess potential protection activity. Preincubation with dendrimers contributed to: increased viability, higher mitochondrial membrane potential, and reduced ROS level in cells. The probable mechanism of cell protection lies in the ability of dendrimers to capture rotenone by encapsulating or binding to its surface groups. The fact that dendrimers have prevention potential is important in the search for new pharmacological strategies against neurodegenerative disorders.

In vitro PAMAM, phosphorus and viologen-phosphorus dendrimers prevent rotenone-induced cell damage.

We have investigated whether polyamidoamine (PAMAM), phosphorus (pd) and viologen-phosphorus (vpd) dendrimers can prevent damage to embryonic mouse hippocampal cells (mHippoE-18) caused by rotenone, which is used as a pesticide, insecticide, and as a nonselective piscicide, that works by interfering with the electron transport chain in mitochondria. Several basic aspects, such as cell viability, production of reactive oxygen species and changes in mitochondrial transmembrane potential, were analyzed. mHippoE-18 cells were treated with these structurally different dendrimers at 0.1µM. A 1h incubation with dendrimers was followed by the addition of rotenone at 1µM, and a further 24h incubation. PAMAM, phosphorus and viologen-phosphorus dendrimers all increased cell viability (reduced cell death-data need to be compared with untreated controls). A lower level of reactive oxygen species and a favorable effect on mitochondrial system were found with PAMAM and viologen-phosphorus dendrimers. These results indicate reduced toxicity in the presence of dendrimers.

[Applications of electromagnetic radiation in medicine]. / Zastosowanie promieniowania elektromagnetycznego w medycynie.

Recent decades have been devoted to the intense search for the response to questions related to the impact of radiation on the human body. Due to the growing fashion for a healthy lifestyle, increasing numbers of works about the alleged dangers of electromagnetic waves and diseases that they cause appeared. However, the discoveries of 20th century, and knowledge of the properties of electromagnetic radiation have allowed to broaden the horizons of the use of artificial sources of radiation in many fields of science and especially in medicine. The aim of this paper is to show that although excessive radiation or high doses are dangerous to the human body, its careful and controlled use, does not pose a threat, and it is often necessary in therapy. The possibility of using ionizing radiation in radiotherapy, isotope diagnostics or medical imaging, and non-ionizing radiation in the treatment for dermatological disorders and cancers will be presented. The unique properties of synchrotron radiation result in using it on a large scale in the diagnosis of pathological states by imaging methods.

Promising low-toxicity of viologen-phosphorus dendrimers against embryonic mouse hippocampal cells.

A new class of viologen-phosphorus dendrimers (VPDs) has been recently shown to possess the ability to inhibit neurodegenerative processes in vitro. Nevertheless, in the Central Nervous Systems domain, there is little information on their impact on cell functions, especially on neuronal cells. In this work, we examined the influence of two VPD (VPD1 and VPD3) of zero generation (G0) on murine hippocampal cell line (named mHippoE-18). Extended analyses of cell responses to these nanomolecules comprised cytotoxicity test, reactive oxygen species (ROS) generation studies, mitochondrial membrane potential (ΔΨm) assay, cell death detection, cell morphology assessment, cell cycle studies, as well as measurements of catalase (CAT) activity and glutathione (GSH) level. The results indicate that VPD1 is more toxic than VPD3. However, these two tested dendrimers did not cause a strong cellular response, and induced a low level of apoptosis. Interestingly, VPD1 and VPD3 treatment led to a small decline in ROS level compared to untreated cells, which correlated with slightly increased catalase activity. This result indicates that the VPDs can indirectly lower the level of ROS in cells. Summarising, low-cytotoxicity on mHippoE-18 cells together with their ability to quench ROS, make the VPDs very promising nanodevices for future applications in the biomedical field as nanocarriers and/or drugs per se.

Mechanism of cationic phosphorus dendrimer toxicity against murine neural cell lines.

The purpose of this manuscript is to study the toxic responses against murine embryonic hippocampal cells (mHippoE-18) and neuroblastoma cells (N2a) to treatment with cationic phosphorus dendrimers (CPD). Two low generations of CPD--generation 2 (G2) and generation 3 (G3)--were applied to cell cultures to monitor events leading to either apoptosis or necrosis. These processes were analyzed using several bioassays, which included the detection of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) alterations, morphology changes, apoptotic and dead cells, cytochrome c (Cyt c) release, caspase 3 activity, DNA fragmentation, as well as changes in cell cycle phases distribution. The results showed that CPD became highly cytotoxic at concentrations above 1 µM and at 0.7 µM in the case of G3 for mHippoE-18 cells. The toxicity was manifested by a pronounced decrease in cell viability, which is correlated with disturbances in cellular activities, such as massive ROS generation. The breakdown of cellular processes leads mainly to the necrotic cell death. Our findings are of high importance in the context of further biomedical studies on CPD.

Viologen-phosphorus dendrimers exhibit minor toxicity against a murine neuroblastoma cell line.

Dendrimers containing viologen (derivatives of 4,4'-bipyridyl) units in their structure have been demonstrated to exhibit antiviral activity against human immunodeficiency virus (HIV-1). It has also recently been revealed that novel dendrimers with both viologen units and phosphorus groups in their structure show different antimicrobial, cytotoxic and hemotoxic properties, and have the ability to influence the activity of cholinesterases and to inhibit α-synuclein fibrillation. Since the influence of viologen-phosphorus structures on basic cellular processes had not been investigated, we examined the impact of such macromolecules on the murine neuroblastoma cell line (N2a). We selected three water-soluble viologen-phosphorus (VPD) dendrimers, which differ in their core structure, number of viologen units and number and type of surface groups, and analyzed several aspects of the cellular response. These included cell viability, generation of reactive oxygen species (ROS), alterations in mitochondrial activity, morphological modifications, and the induction of apoptosis and necrosis. The MTT assay results suggest that all of the tested dendrimers are only slightly cytotoxic. Although some changes in ROS formation and mitochondrial function were detected, the three compounds did not induce apoptosis or necrosis. In light of these results, we can assume that the tested VPD are relatively safe for mouse neuroblastoma cells. Although more research on their safety is needed, VPD seem to be promising nanoparticles for further biomedical investigation.

Viologen-Phosphorus Dendrimers Inhibit α-Synuclein Fibrillation.

Inhibition of α-synuclein (ASN) fibril formation is a potential therapeutic strategy in Parkinson's disease and other synucleinopathies. The aim of this study was to examine the role of viologen-phosphorus dendrimers in the α-synuclein fibrillation process and to assess the structural changes in α-synuclein under the influence of dendrimers. ASN interactions with phosphonate and pegylated surface-reactive viologen-phosphorus dendrimers were examined by measuring the zeta potential, which allowed determining the number of dendrimer molecules that bind to the ASN molecule. The fibrillation kinetics and the structural changes were examined using ThT fluorescence and CD spectroscopy. Depending on the concentration of the used dendrimer and the nature of the reactive groups located on the surface, ASN fibrillation kinetics can be significantly reduced, and even, in the specific case of phosphonate dendrimers, the fibrillation can be totally inhibited at low concentrations. The presented results indicate that viologen-phosphorus dendrimers are able to inhibit ASN fibril formation and may be used as fibrillar regulating agents in neurodegenerative disorders.

Dendrimers--revolutionary drugs for infectious diseases.

Over recent years innovative nanomolecules in a form of dendrimers have been gaining increasing interest. These compounds can be designed and modified in many ways giving a molecule which meets required expectations. For this reason dendrimers are the object of intensive studies in many fields of nanoscience including one of the most thriving--biomedicine. Numerous studies provide evidence that some dendrimers exhibit activities against many species/strains of viruses, bacteria, fungi, and prions. These types of dendritic nanostructures which are distinguished by antipathogenic properties and low cytotoxicity to eukaryotic cells may be potentially applied in medicine as novel drugs for various infectious diseases, especially those which are persistent, marked by high mortality rate, or untreatable. Dendrimers can exert their effect via different mechanisms of action, which are, in most cases, related to multivalency of the nanomolecule. The application of dendrimers is likely to be a breakthrough in prevention and treatment of infectious diseases which still beset humanity and may significantly improve the quality of people's life.

Phosphorus-containing dendrimers against α-synuclein fibril formation.

The aim of this work was to study the effect of phosphorus-containing dendrimers (generations G3 and G4) on the fibrillation of α-synuclein (ASN). The inhibition of fibril formation (filamentous and aggregates) is a potential therapeutic strategy for neurodegenerative disorders such as Parkinson's and other motor disorder neurodegenerative diseases. The interaction between phosphorus-containing dendrimers and ASN was studied by fluorescence spectroscopy. The decrease in the fluorescence intensity of intrisinic tyrosine was the most marked change in the fluorescence intensity observed upon addition of dendrimers. Furthermore, the effect of dendrimers on ASN fibril formation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by fluorescence assays using the dye thioflavin T (ThT). The results showed that phosphorus-containing dendrimers G3 and G4 inhibited fibril formation, when they were used in the ASN/dendrimer ratios 1:0.1 and 1:0.5. However, the higher concentrations of dendrimers did not show this effect.

Interaction of polyamidoamine (PAMAM) succinamic acid dendrimers generation 4 with human serum albumin.

Dendrimers, a relatively new group of highly branched three dimensional polymers, are intensively investigated to use them in biomedical and physicochemical sciences. Their specific architecture gives them the ability to interact with many different types of molecules. In our studies the interaction between PAMAM succinamic acid dendrimers generation 4 (PAMAM-SAH G4) and human serum albumin (HSA) was examined. Experiments showed that a single molecule of a HSA can bind approximately 6 particles of dendrimers. The fluorescence studies demonstrated that dendrimers lead to a decrease in protein fluorescence but changes in fluorescence anisotropy were not observed. Alterations in the spectrum of circular dichroism indicated changes in the secondary protein structure. The results clearly show that this generation of dendrimers possesses a strong ability to interact with human serum albumin.

PAMAM G4 dendrimers affect the aggregation of α-synuclein.

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.

Interaction between PAMAM-NH2 G4 dendrimer and 5-fluorouracil in aqueous solution.

The formation equilibrium of poly(amidoamine) dendrimer (PAMAM-NH2 G4) complex with an oncologic drug such as 5-fluorouracil (5-FU) in water at room temperature was examined. Using the results of the drug solubility in dendrimer solutions and the method of equilibrium dialysis, the maximal number of drug molecules in the dendrimer-drug complex and its equilibrium constant were evaluated. Solubility results show that PAMAM-NH2 G4 dendrimer can transfer tens 5-fluorouracil molecules in aqueous solution. The number of active sites in a dendrimer macromolecule being capable of combining the drug, determined by the separation method, amounts to n=30 ± 4. The calculated equilibrium constant of the 5-FU-active site bonding is equal to K=(400 ± 120).

Effect of poly(amido)amine (PAMAM) G4 dendrimer on heart and liver mitochondria in an animal model of diabetes.

Diabetes-induced injury related to hyperglycaemia is associated with impaired function of mitochondria. Regardless of their cytotoxicity, PAMAM [poly(amido)amine] G4 dendrimers lower plasma glucose and suppress long-term markers of diabetic hyperglycaemia in experimental diabetes. In the present study, we aimed at verifying whether such modulatory effects of PAMAM G4 (0.5 micromol/kg of body weight daily for 60 days) may contribute to improved respiration in heart and liver mitochondria from streptozotocin-diabetic rats. PAMAM G4 alleviated long-term markers of hyperglycaemia and reduced blood and tissue lipophilic antioxidants in diabetic animals, but did not restore mitochondrial function. In hearts, but not livers, dendrimers further reduced respiratory function and oxidative phosphorylation. Thus ameliorating effects of PAMAM G4 on glycation and glycoxidation in experimental diabetes are not sufficient to restore the impaired mitochondrial function in diabetes.

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax.

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe3+ ions. Erythrocytes were exposed to various concentrations of CuSO4, Fe(NO3)3, and K3Fe(CN)6 for up to 5h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO3)3. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO4 increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 microM. This effect was decreased by potassium ferricyanide and Fe(NO3)3 only after 1 and 3h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 microM CuSO4 as well as 20 microM Fe(NO3)3 and 10 mM K3Fe(CN)6 induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K3Fe(CN)6. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO3)3.

Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro.

Genistein-8-C-glucoside (G8CG) belongs to natural isoflavones phytoestrogens, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants, with possible anticarcinogenic effects in various in vitro systems and in vivo animal models. We used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.) to study its cytotoxic and genotoxic effects on mouse embryonic fibroblast (line NIH 3T3). The MTT assay to assess cytotoxicity and comet assay for the detection of DNA damage were used. The cells were exposed to various concentrations of genistein-8-C-glucoside (2.5-110 microM) and hydrogen peroxide (5-90 microM). The effect of G8CG alone or in combination with H2O2 was determined. G8CG at concentrations > 20 microM significantly reduced cell viability and induced DNA damage. In contrast, lower concentrations of (2.5-10 microM) G8CG showed antioxidant properties against H2O2-induced DNA damage with no associated toxicity.

The interaction between PAMAM G3.5 dendrimer, Cd2+, dendrimer-Cd2+ complexes and human serum albumin.

The interactions between PAMAM G3.5 dendrimer, Cd2+, a complex of "PAMAM G3.5 dendrimer-Cd2+" and human serum albumin were studied by equilibrium dialysis, fluorescence quenching, fluorescence anisotropy and zeta-potential. It was found that in water one molecule of PAMAM 3.5 dendrimer can bind 38+/-9 cadmium ions with Kb=1.3+/-0.13 x 10(3). The calculated Stern-Volmer constants of albumin fluorescence quenching by Cd2+, G3.5 and the "PAMAM G3.5-Cd2+" complex were 2.2+/-0.2, 1.6+/-0.3 and 1.4+/-0.1(mmol/l)(-1), respectively. The data from the fluorescence and zeta-potential studies show that the "PAMAM G3.5-Cd2+" complex interacted much less strongly with human serum albumin than the pure dendrimer or Cd2+.

PAMAM G4 dendrimers lower high glucose but do not improve reduced survival in diabetic rats.

For nearly a decade poly(amidoamine) (PAMAM) dendrimers G4 were claimed unnegligible cytotoxic agents. Here we monitored whether in vivo cytotoxic effect of PAMAM G4 (0.5 micromol kg(-1) day(-1)) may be compromised by its ameliorating effect on severe hyperglycaemia in chronic streptozotocin-diabetic Wistar rats. PAMAM G4 significantly reduced the 60-day overall survival in long-term experimental diabetes: treated animals were 6.7 times more likely to die than control animals (p<0.025). PAMAM G4 significantly reduced numerous biochemical parameters in blood, including glucose, glycated haemoglobin or protein oxidation, cholesterol and triglycerides, but apparently unchanged plasma insulin peptide C. Terminal blood glucose in PAMAM-treated animals was significantly higher in survivors, pointing to the possible preventive role of glycation in reducing of PAMAM G4 cytotoxicity. Our results provide the first in vivo evidence that PAMAM G4 is able to lower plasma glucose and suppress long-term markers of diabetic hyperglycaemia. Nevertheless, this beneficial influence cannot override PAMAM G4 cytotoxic effects in the increased mortality of streptozotocin-diabetic rats.

Preliminary biological evaluation of poli(amidoamine) (PAMAM) dendrimer G3.5 on selected parameters of rat liver mitochondria.

Polyamidoamino (PAMAM) dendrimer of generation 3.5 (G3.5) specific interactions with rat liver mitochondria were studied. Selected parameters of mitochondria (transmembrane potential and uptake of Ca2+ ions) were investigated after the exposure to G3.5 used at the concentration of 10, 30 and 50 microM and Ca2+ ions used at 1mM. The times of preincubation of isolated liver mitochondria with dendrimer were 5, 15 and 30 min at room temperature. The mitochondrial membrane potential was monitored spectrofluorimetrically by fluorescence quenching of Rhodamine 123 (Rh 123). The changes in calcium homeostasis upon dendrimer exposure were detected using flow cytometric analysis with Fluo-3. On the one hand the obtained results revealed an impact of the tested chemical on rat liver mitochondrial function. We found that dendrimer G3.5 in a concentration above 10 microM contributes to the reduction in the transmembrane potential and hinders in the influx of Ca2+ ions to mitochondria added externally, or accelerates their efflux from mitochondria. The most effective preincubation time was observed to be 15 min for all tested concentrations. On the other hand the combined treatment of dendrimer G3.5 with Ca2+ demonstrated the protective effect of G3.5 against mitochondrial depolarization caused by calcium ions. These preliminary studies suggest that tested dendrimer can significantly affect the mitochondria and modulate their functionality.