Studying complexes between PPI dendrimers and Mant-ATP.

The aim of this study was to investigate the interactions between poly(propylene imine) (PPI) dendrimers and 2'-/3'-O-(N'-methylanthraniloyl)-ATP (Mant-ATP). Mant-ATP was used as a model molecule. Purine and pyrimidine nucleoside analogues are antimetabolites commonly used in therapy for cancer. Drug molecules can complex with dendrimers in two ways: therapeutic agents may be attached in dendrimer interior or bind to functional groups on the surface. Drugs attached to nanoparticles are characterized by improved solubility, pharmacokinetics and stability. Here, we have used poly(propylene imine) dendrimers of the 4th and 5th generations (PPI G4 and PPI G5) with primary amino surface groups partially modified with maltose (PPI-m) or without modification (PPI). We assessed the efficiency of complex formation in relation to dendrimer generation, pH of solution and the type of dendrimer used. A double fluorimetric titration method was used to estimate the binding constant (K b ) and the number of binding centers per molecule of the binding agent (n).

Glycodendrimers as new tools in the search for effective anti-HIV DC-based immunotherapies.

Dendritic cells (DC), which play a major role in development of cell-mediated immunity, represent opportunities to develop novel anti-HIV vaccines. Dendrimers have been proposed as new carriers to ameliorate DC antigen loading and in this way, we have determined the potential use of maltose decorated neutrally and positively charged G4 glycodendrimers. Thus, immunostimulatory properties of these glycodendrimers on human DC were evaluated in the context of HIV infection. We have demonstrated that DC treated with glycodendrimers were fully functional with respect to viability, maturation and HIV-derived antigens uptake. Nevertheless, iDC and mDC phenotypes as well as mDC functions such as migration ability and cytokines profile production were changed. Our results showed the potential carrier properties of glycodendrimers to activate the immune system by the way of DC stimulation. This is the first study for exploring the use of maltose-functionalized dendrimers-peptides complexes as a potential DC-based vaccine candidate. FROM THE CLINICAL EDITOR: In this paper, maltose-functionalized dendrimer-peptide complexes are demonstrated to activate the immune system by way of dendritic cell (DC) stimulation. DC vaccination using this methodology may be applicable to a variety of conditions, including infections and potentially cancer.

Neurons and stromal stem cells as targets for polycation-mediated transfection.

Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.

Dendrimers in gene transfection.

Dendrimers are a new class of nanocomposite materials. They are branching polymers whose structure is formed by monomeric subunit branches diverging to all sides from a central nucleus. The type of nucleus, attached monomers, and functional groups can be chosen during synthesis, which produces dendrimers of definite size, shape, density, polarity, branch mobility, and solubility. This review deals with problems of dendrimer molecular structures and capability of in vitro, in vivo, ex vivo, and in situ transfection of genetic material. Advantages and shortcomings of different types of dendrimers in this respect are discussed.

Interactions between PAMAM dendrimers and gallic acid molecules studied by spectrofluorimetric methods.

Interactions between gallic acid molecules and different types of polyamidoamine (PAMAM) dendrimers with modified surfaces were studied by spectrofluorimetric methods. Changes in fluorescence intensity of gallic acid and in a position of spectrum were monitored. It was found that the extent of gallic acid incorporation into dendrimers depends on a type of a dendrimer.

Melatonin directly scavenges free radicals generated in red blood cells and a cell-free system: chemiluminescence measurements and theoretical calculations.

Melatonin, a pineal secretory product, has properties of both direct and indirect powerful antioxidant. The aim of the present study was to compare the radical-scavenging, structural and electronic properties of melatonin and tryptophan, precursor of melatonin. Using the alkoxyl- and peroxyl radical-generating systems [the organic peroxide-treated human erythrocytes and a cell-free system containing the azo-initiator 2,2'-azobis(2-amidinopropane)dihydrochloride], we evaluated the radical-scavenging effects of melatonin and tryptophan. Melatonin rather than tryptophan at concentrations of 100-2000 microM markedly inhibited membrane lipid peroxidation in human erythrocytes treated with organic hydroperoxide as well as radical-induced generation of luminol-dependent chemiluminescence. The apparent Stern-Volmer constants for inhibition of membrane lipid peroxidation by melatonin and tryptophan were estimated to be (0.23+/-0.05) x 10(4) M(-1) and (0.02+/-0.005) x 10(4) M(-1), respectively. The apparent Stern-Volmer constants for inhibition of azo-initiator-derived peroxyl radical generation by melatonin and tryptophan were determined to be (0.42+/-0.05) x 10(4) M(-1) and (0.04+/-0.01) x 10(4) M(-1), respectively. The structural and electronic properties of melatonin and its precursor, tryptophan, were determined theoretically by performing semi-empirical and ab initio calculations. The high radical-scavenging properties of melatonin may be explained by the high surface area value and high dipole moment value. From the thermodynamic standpoint, based on our calculations, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), was the most stable end oxidative product of melatonin.

Effect of dendrimers on pure acetylcholinesterase activity and structure.

The effect of polyamidoamine (PAMAM) dendrimers on activity and fluorescence of pure acetylcholinesterase (EC 3.1.1.7.) was studied. It has been shown that all dendrimers studied decreased the enzymatic activity of acetylcholinesterase. This effect depended on the type of dendrimers. The data on the intrinsic fluorescence have shown that the dendrimers changed acetylcholinesterase conformation and the strongest effect was induced by PAMAM G3.5 dendrimer.

DSC studies on interactions between low molecular mass peptide dendrimers and model lipid membranes.

It has recently been shown that a newly synthesized peptide dendrimers possess antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria as well as against fungal pathogens (Candida albicans) [Klajnert, B., Janiszewska, J., Urbanczyk-Lipkowska, Z., Bryszewska, M., Shcharbin, D., Labieniec, M., 2006. Biological properties of low molecular mass peptide dendrimers. Int. J. Pharm. 309, 208-217]. To extend our knowledge about their impact on biological systems, interactions between a group of low molecular mass lysine based dendrimers and model lipid bilayers were examined by differential scanning calorimetry (DSC). Conformational stability of dendrimers in 5-85 degrees C temperature range was confirmed by circular dichroism measurements (CD). The dendrimer structure has been shown to play an important role in interactions with the membranes. A two-step mechanism of dendrimer-bilayer interactions was proposed. The first step involves electrostatic attractions between dendrimers and polar lipid heads, while the second one is a result of hydrophobic interactions between acyl chains and arms of dendrimers. While one dendrimer did not interact with the membrane, another with long hydrophobic arms significantly perturbed the membrane. Nevertheless, for all tested dendrimers the main transition in DSC scans was retained that indicates that these compounds at the tested concentrations did not cause the loss of membrane integrity.

Biological properties of low molecular mass peptide dendrimers.

A series of new, low molecular mass, lysine-based peptide dendrimers with varying distribution of cationic and aromatic groups in the structure were synthesized. They expressed antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria as well as against fungal pathogens (Candida albicans). Their cytotoxic, haematotoxic, and genotoxic effects were studied. It appears that degree of branching and steric distribution and types of hydrophobic (aromatic) groups and cationic centres are important components of dendrimeric structure and influence both antimicrobial potency and toxicity. Such 3D structure of our dendrimers mimics that of the natural antimicrobial peptides and can be achieved by application of dendrimer chemistry.

The response of Na+/K+ -ATPase of human erythrocytes to green laser light treatment.

The objective of this study was to investigate the response of Na(+)/K(+)-ATPase of human erythrocytes to green laser irradiation. Effects of green laser light of fluences 9.5-63.3 J.cm(-2) and merocyanine 540-mediated laser light treatment were studied. Isolated erythrocyte membranes (protein concentration of 1 mg/ml) were irradiated by Nd:YAG laser (532 nm, 30 mW) and then incubated in a medium with 2 mM ATP for 30 min. Activity of ATPase was determined colorimetrically by measuring the colored reaction product of liberated inorganic phosphate and malachite green at 640 nm. Contribution of Na(+)/K(+)-ATPase to overall phosphate production was determined using ouabain. A positive effect of green laser light on Na(+)/K(+)-ATPase activity was observed. The dependence of enzymatically liberated inorganic phosphate on light fluence showed a linear correlation (R(2)=0.96, P=0.0005) for all fluences applied (9.5-63.3 J.cm(-2)). On the other hand, MC 540-mediated phototreatment caused a suppression of enzyme activity.

Effects of sugar alcohols and disaccharides in inducing the hexagonal phase and altering membrane properties: implications for diabetes mellitus.

A number of sugars lowered the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. Disaccharides had the greatest effect followed by sugar alcohols. The monosaccharides, glucose and galactose had no effect on this phase transition temperature. The sugars promoted vesicle leakage only under conditions where the lipid was near its hexagonal phase transition temperature. Leakage from lipids in the bilayer state was inhibited by the sugars. Polyols, such as sorbitol, promote hexagonal phase formation and alter membrane permeability. These membrane effects may contribute to the damage caused by sorbitol accumulation in certain tissues of diabetic patients.

Oxidative damage of Chinese hamster fibroblasts induced by t-butyl hydroperoxide and by X-rays.

The aim of this study was to investigate the mechanism(s) of X-ray-mediated cell damage in comparison to mechanism(s) of organic hydroperoxide cytotoxicity and to find the main targets for the two different kinds of cell inactivation. Damage of Chinese hamster fibroblasts induced by tert-butyl hydroperoxide (t-BHP) or X-irradiation was measured by the colony-formation assay and the average single colony volume. DNA double-strand breaks (dsb) were determined by constant-field gel electrophoresis. The contents of peroxides, of SH-groups and the size of inactivated cells were tested for oxidative modifications.Oxidative damage of fibroblasts induced by t-BHP or by X-rays inhibits cell proliferation. Simultaneously, irradiation causes an increase of DNA dsb with the dose, while incubation with t-BHP yields only a very few DNA dsb. Neither chemically induced oxidation nor irradiation significantly changed the amount of membrane lipid peroxides. Oxidation with t-BHP but not irradiation leads to a loss of the membrane SH-groups and to an increase of cell diameter. The similar decrease of cell proliferation can be caused by DNA dsb without detectable membrane damage (X-radiation) as by membrane damage with nearly no DNA dsb (chemically induced oxidative stress).

Membrane effects of nitrite-induced oxidation of human red blood cells.

The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.

Reduced membrane fluidity in platelets from diabetic patients.

Platelets from diabetic patients are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function, and reduced membrane fluidity in cholesterol-enriched platelets is associated with platelet hypersensitivity to agonists, including thrombin. Decreased membrane fluidity of these platelets is attributed to an increased cholesterol-phospholipid molar ratio in platelet membranes. We examined the response of platelets from diabetic subjects to thrombin, platelet membrane fluidity, and platelet cholesterol-phospholipid molar ratio. Twelve poorly controlled diabetic subjects were compared with 12 age- and sex-matched control subjects. In response to a low concentration of thrombin, mean values for release of [14C]serotonin from washed prelabeled platelets were not significantly different between diabetic and control subjects, but in 8 of 12 diabetic subjects, the release response was greater than in their paired control subjects. Mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5-hexatriene-labeled platelets prepared from diabetic subjects were significantly greater than in control subjects; this indicates a decreased membrane fluidity in platelets from diabetic subjects. Total or very-low-density (VLDL), low-density (LDL), or high-density (HDL2, HDL3) lipoprotein cholesterol concentrations in plasma were not significantly different between groups; however, the ratio of VLDL + LDL to HDL2 + HDL3 was significantly greater in diabetic than in control subjects. There was no difference in the total platelet cholesterol-phospholipid molar ratio between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial alterations induced by 532 nm laser irradiation.

Mitochondrial alterations were monitored after low power green laser (532 nm, 30 mW) irradiation in the case of whole cells (B-14) and isolated mitochondria (from Wistar rat heart). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay products were significantly higher (by 8%) in irradiated B-14 cells as compared to non-irradiated controls. Mitochondrial transmembrane potential of B-14 cells, measured by means of a fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), significantly increased (by 13%) after exposure to green laser irradiation. Another MTT assay was used for isolated mitochondria suspensions in order to examine the effect of green laser irradiation on stimulation of processes related to oxidative phosphorylation. It revealed 31.3%-increase in MTT assay products in irradiated mitochondria as compared to controls. Laser irradiation of isolated mitochondria suspension did not significantly change 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, indicating that mitochondrial membrane fluidity was not affected by laser light. Fluorescence emission spectra of irradiated as well as non-irradiated mitochondria suspensions showed fluorescence maximum at 635 nm, corresponding to emission of Protoporphyrin IX, which was significantly lower (by 20.7%) in irradiated sample.

Hypochlorous acid damages erythrocyte membrane proteins and alters lipid bilayer structure and fluidity.

Treatment of human erythrocyte membranes with active forms of chlorine (hypochlorous acid and chloramine T) resulted in a concentration-dependent inhibition of the membrane Na(+), K(+)- and Mg(2+)-ATPases. Membrane protein thiol group oxidation was consistent with inactivation of enzymes and preceded oxidation of tryptophan residues and chloramine formation. Erythrocyte exposure to hypochlorous acid led to complex changes of cell membrane rigidity and cell morphological transformations: cell swelling, echinocyte formation, and haemolysis. The inhibition of ion pump ATPases of human erythrocyte membranes may be due to direct oxidation of essential residues of enzyme (thiol groups) and structural rearrangement of the membrane.

Association between the glycation of erythrocyte membrane proteins and membrane fluidity.

Erythrocyte membrane ghosts prepared from normal individuals were incubated in vitro at different glucose concentrations for 72 h. Incubation with increasing concentrations of glucose resulted in increased glycation (nonenzymatic glycosylation) of membrane proteins and a decrease of erythrocyte membrane fluidity measured by the use of the fluorescent label pyrene. It would therefore seem likely that the changes in erythrocyte membrane fluidity previously reported in diabetic subjects (1-3) are related to in vivo glycation of membrane proteins as well as to changes in lipid composition, as was previously suggested (2).

Morphological changes of human erythrocytes induced by cholesterol sulphate.

OBJECTIVES: Morphological alterations of human erythrocytes induced by cholesterol sulphate (5-cholesten-3 beta-ol sulphate, CS) were studied. DESIGN AND METHODS: Influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy was studied. RESULTS: In isotonic medium CS induces erythrocyte size and shape changes in dose-and time-dependent manner. Incubation (in vitro) of erythrocytes with CS concentrations from 4 x 10(-5) mol/dm3 to 8 x 10(-5) mol/dm3 led to a progressive sphero-echinocitic shape transformation accompanied by a cell size decrease. In contrast to this, for CS content equal to 1 x 10(-5) mol/dm3 the maintenance of the normal biconcave shape of red blood cells was observed. CONCLUSIONS: The results suggest that CS, similarly to numerous evaginating amphiphilic agents, induces a transformation of the erythrocyte normal discoid shape to echinocytic form. This effect may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of CS incorporated into the membrane. The echinocytic shape transformation of erythrocytes indicated that CS intercalates in the outer hemileaflet of the lipid bilayer leading to membrane externalization.

Estimation of cholesterol sulphate in blood plasma and in erythrocyte membranes from individuals with Down's syndrome or diabetes mellitus type I.

OBJECTIVES: Plasma and erythrocyte membrane cholesterol sulphate (CS) were measured in patients suffering from diabetes and Down's syndrome. DESIGN AND METHODS: The procedure for separation and determination of CS comprised HPTLC (high-performance thin-layer chromatography) and densitometry. RESULTS: The mean plasma and RBC membranes CS concentrations (+/- SD) of the control group (n = 16) was 188 +/- 47 micrograms/dL and 343 +/- 57 micrograms/10(12) RBC, respectively. In 15 patients with diabetes and 12 Down's syndrome patients substantially higher CS levels were found (diabetes: plasma-348 +/- 60 micrograms/dL; RBC membranes-646 +/- 113 micrograms/10(12) RBC; Down's syndrome: plasma-245 +/- 54 micrograms/dL; RBC membranes 427 +/- 74 micrograms/10(12) RBC). Analysis of variance and multiple comparison (Newman-Keuls test) show statistically significant differences between all samples both for erythrocytes, F(2.41) = 52.24, p < 0.05, and plasma, F(2.41) = 34.92, p < 0.05. CONCLUSIONS: It is postulated that differences in CS levels may contribute to changes of erythrocyte properties in these pathological states.

Dendrimers: properties and applications.

Dendrimers are a new class of polymeric materials. They are highly branched, monodisperse macromolecules. The structure of these materials has a great impact on their physical and chemical properties. As a result of their unique behaviour dendrimers are suitable for a wide range of biomedical and industrial applications. The paper gives a concise review of dendrimers' physico-chemical properties and their possible use in various areas of research, technology and treatment.