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).

Molecular properties of lysine dendrimers and their interactions with Aß-peptides and neuronal cells.

Prevention of amyloidosis by chemical compounds is a potential therapeutic strategy in Alzheimer's, prion and other neurodegenerative diseases. Regularly branched dendrimers and less regular hyperbranched polymers have been suggested as promising inhibitors of amyloid aggregation. As demonstrated in our previous studies, some widely used dendrimers (PAMAM, PPI) could not only inhibit amyloid aggregation in solution but also dissolve mature fibrils. In this study we have performed computer simulation of polylysine dendrimers of 3rd and 5th generations (D3 and D5) and analysed the effect of these dendrimers and some hyperbranched polymers on a lysine base (HpbK) on aggregation of amyloid peptide in solution. The effects of dendrimers on cell viability and their protective action against Aß-induced cytotoxicity and alteration of K+channels was also analysed using human neuroblastoma SH-SY5Y cells. In addition, using fluorescence microscopy, we analysed uptake of FITC-conjugated D3 by SH-SY5Y cells and its distribution in the brain after intraventricular injections to rats. Our results demonstrated that dendrimers D3 and D5 inhibited amyloid aggregation in solution while HpbK enhanced amyloid aggregation. Cell viability and patch-clamp studies have shown that D3 can protect cells against Aß-induced cytotoxicity and K+channel modulation. In contrast, HpbK had no protective effect against Aß. Fluorescence microscopy studies demonstrated that FITC-D3 accumulates in the vacuolar compartments of the cells and can be detected in various brain structures and populations of cells after injections to the brain. As such, polylysine dendrimers D3 and D5 can be proposed as compounds for developing antiamyloidogenic drugs.

Cytotoxicity and genotoxicity of cationic phosphorus-containing dendrimers.

Cationic phosphorus-containing dendrimers (CPDs) are a class of highly-branched polymers with potential medical relevance. However, little is known about CPD modes of interactions with cell and its components, including DNA. In the present work we investigated cytotoxicity and genotoxicity of CPDs generation 3 and 4 (CPD G3 and CPD G4) in human mononuclear blood cells, A549 human cancer cells and human gingival fibroblasts (HGFs). CPD G3 and CPD G4 at concentrations up to 10 µM induced a concentration-dependent decrease in cell viability as assessed by flow cytometry. Both compounds did not induce breaks in isolated DNA as evaluated by the plasmid relaxation assay but they induced DNA cross-links in the cells, as examined by comet assay. CPD G3 and 4 induced slight perturbations in the cell cycle leading to a decrease in the G2/M cell population accompanied by an increase in the S cell population. Upon treatment with CPDs, the cells showed changes in their morphology, including loss of cell attachment, disruption of cell membrane and nucleus condensation. Our results indicate that CPD G3 and G4 are cytotoxic and genotoxic for the assorted human cells. Therefore, CPDs may form stable complexes with DNA and interfere with cellular processes.

Dendrimers in photodynamic therapy.

Photodynamic therapy (PDT) is a promising approach to treat certain types of cancer. PDT was proposed as a useful oncology tool more than 30 years ago but it has limitations. The success of PDT depends predominantly on photosensitizers and development of an effective second generation is continuing. Dendrimers possess architecture suitable for incorporating specific functional moieties and are a promising venue for further investigations. This review describes the use of dendrimers in PDT and how they can aid in overcoming obstacles encountered during PDT.

Highly organized self-assembled dendriplexes based on poly(propylene imine) glycodendrimer and anti-HIV oligodeoxynucleotides.

Dendrimers are artificial polymeric macromolecules which are widely considered to be a promising tool for future gene therapy applications. They have been used as efficient delivery vehicles for antisense oligonucleotides targeting the interior of cells. We demonstrate that dendriplexes formed from anti-HIV oligodeoxynucleotides ANTI-TAR, GEM91, and SREV in complex with generation 4 maltose (PPI-Mal G4) and maltotriose (PPI-Mal-III G4) modified poly(propylene imine) dendrimers are able to self-assemble into highly organized 1D and 3D nanostructures. The resulting nanostructures were characterized by fluorescence methods, laser Doppler electrophoresis, dynamic light scattering (DLS), atomic force microscopy (AFM) and molecular modeling. The results show that ANTI-TAR and GEM 91 dendriplexes self-assemble into fibrils with length scales up to several hundreds of nm. SREV, on the contrary, forms quadrilateral- like 3D nanostructures. A good correlation between the various experimental methods and molecular modeling indicates the formation of those nanostructures in solution. Space symmetry of the oligonucleotides and the resulting dendriplex monomeric units are probably the most important factors which influence the way of self-assembling.

Kinetics of amyloid and prion fibril formation in the absence and presence of dense shell sugar-decorated dendrimers.

The aggregation behavior of the amyloid peptide Aß(1-28) and the prion peptide PrP(185-208) - both responsible for neurodegenerative disorders - was analyzed in the absence and in the presence of poly(propylene imine) (PPI) dendrimers at generation 5 (G5) with a dense shell of maltose and maltotriose units. Thioflavin T (ThT) fluorescence assay and circular dichroism (CD) experiments indicated that fibril formation is enhanced at low dendrimer concentration, while it is prevented at relatively high dendrimer concentrations. Computer aided EPR analysis by means of the selected spin probe 4-octyl-dimethylammonium,2,2,6,6-tetramethyl-piperidine-1-oxyl bromide (CAT8) further demonstrated this behavior, but also provided detailed information on the mechanism of fibril formation and on the different behavior of the differently decorated dendrimers. The CAT8 radicals were progressively trapped at the peptide interphase when peptide aggregates were formed, also monitoring pre-fibrillar structures. At later time, a phase separation of the CAT8 radicals monitors the formation of further supramolecular structures where the probes become squeezed among fibrillar aggregates. The addition of small amounts of dendrimers promotes the formation of peptide fibrils breaking them and providing a larger amount of ends that serve as sites of replications. Conversely, a high amount of dendrimers allows the peptides to well separate from each other such preventing their aggregation. EPR results also indicate that the perturbation played by PPI(G5)-Maltose are more effective onto PrP(185-208) than onto Aß(1-28), while PPI(G5)-Maltotriose is less effective towards PrP(185-208) in both promoting aggregation and preventing it by changing the dendrimer concentration. These results provide useful information about the mechanism and interactions which regulate the ability of macromolecules like the dendrimers to favor, prevent or cure neurodegenerative diseases.

Mechanism of neuroprotection of melatonin against beta-amyloid neurotoxicity.

The main component of senile plaques in Alzheimer's disease (AD), aggregated amyloid beta peptide (ßA), is neurotoxic and implicated in AD pathology. Melatonin is a hormone secreted from the pineal gland, levels of which are decreased in aging, particularly in AD subjects. This hormone is known to possess neuroprotective properties against ßA toxicity in vivo, but the mechanism of protection remains controversial. In cultures of mixed neurones and astrocytes, we find that melatonin is protective against neuronal and astrocytic death induced by aggregated full length ßA 1-40 and the fragments ßA 25-40 and ßA 1-28. Melatonin had no effect on the process of fibrillation of ßA and did not alter ßA-induced calcium signalling in astrocytes, but did significantly reduce the rate of ßA-induced reactive oxygen species production and also protected astrocytes against the mitochondrial depolarisation. Thus, scavenging of reactive oxygen species by melatonin appears to be the primary effect of melatonin in protecting neurones and astrocytes against ßA toxicity.

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.

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.

Molecular interactions of dendrimers with amyloid peptides: pH dependence.

The formation of amyloid plaques is a key pathological event in neurodegenerative disorders, such as prion and Alzheimer's diseases. Dendrimers are considered promising therapeutic agents in these disorders. In the present work, we have studied the effect of polypropyleneimine dendrimers on the formation of amyloid fibrils as a function of pH in order to gain further insight in the aggregation mechanism and its inhibition. Amyloid fibrils from prion peptide PrP 185-208 and Alzheimer's peptide Abeta 1-28 were produced in vitro, and their formation was monitored using the dye thioflavin T (ThT). The results showed that the level of protonation of His, Glu, and Asp residues is important for the final effect, especially at low dendrimer concentration when their inhibiting capacity depends on the pH. At the highest concentrations, dendrimers were very effective against fibril formations for both prion and Alzheimer's peptides.

Influence of dendrimer's structure on its activity against amyloid fibril formation.

Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer's diseases. Highly branched, globular polymers-dendrimers-are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185-208 and the Alzheimer's peptide Abeta 1-28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer-peptide interaction mechanism is presented based on the dendrimers' molecular structure.

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.

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.

Use of a spectrofluorimetric method to monitor changes of human serum albumin thermal stability in the presence of polyamidoamine dendrimers.

Polyamidoamine (PAMAM) dendrimers' impact on human serum albumin thermal stability was investigated. Thermal denaturation profiles were determined from changes in the intrinsic fluorescence intensity. In the presence of dendrimers the shift of a denaturation temperature toward higher values was observed. It indicates a slight increase of protein stability upon dendrimers treatment.

PAMAM dendrimers and model membranes: differential scanning calorimetry studies.

Dendrimers attract much attention as potential drug and gene carriers for intracellular delivery. From this point of view, it is crucial to extend our knowledge about their interactions with membranes. The influence of polyamidoamine (PAMAM) dendrimers on the thermotropic behavior of DPPC multilamellar vesicles and DMPC small unilamellar vesicles was examined by differential scanning calorimetry. We used three types of PAMAM dendrimers to determine how a dendrimer structure determines interactions with liposomes. We show that the strength of interactions depends on both the dendrimers' structure and degree of hydrophobicity. A model for the interaction of each type of dendrimer with liposomes was proposed.

The effect of PAMAM dendrimers on human and bovine serum albumin at different pH and NaCl concentrations.

The effect of PAMAM G3.5, PAMAM G4 and PAMAM-OH G4 dendrimers on human and bovine serum albumins has been studied by fluorescence spectroscopy at different pH and ionic strength. It has been shown that the interactions between dendrimers and proteins depend on pH and the efficiency of interactions can be regulated by changing pH. The maximal pH dependence was observed for interactions between albumins and PAMAM G4 dendrimer. At physiological pH all dendrimers affect proteins in the maximum degree. Dendrimers had no effect on N-F and N-B transitions of albumins. The effect of dendrimers on HSA was smaller than for BSA. The increase of NaCl concentration led to a decrease of interactions between dendrimers and proteins.

Dendrimer interactions with hydrophobic fluorescent probes and human serum albumin.

The interactions between three types of polyamidoamine dendrimers (with anionic, cationic, and neutral charge on a surface) and fluorescent dye 1-anilinonaphthalene-8-sulfonate (ANS) were studied. Double fluorimetric titration method was employed to estimate a binding constant and the number of binding centers. As fluorescent probes can serve as models of toxin molecules, dendrimers, and human serum albumin (HSA) abilities to bind ANS were compared. In the presence of HSA and dendrimers, ANS located both in HSA and in dendrimers, but the interactions between ANS and HSA were stronger.

The effect of polyamidoamine dendrimers on human erythrocyte membrane acetylcholinesterase activity.

Polyamidoamine (PAMAM) dendrimers impact on activity of acetylcholinesterase was studied. It has been shown that dendrimers induce a biphasic effect: depending on their concentrations they increase or decrease enzyme activity. It may be due to two types of interactions: direct--between dendrimers and the enzyme; indirect--via a modification of the physical state of membrane phospholipids affecting the acetylcholinesterase.

The effect of Pycnogenol on the erythrocyte membrane fluidity.

In the present study, the in vitro effect of polyphenol rich plant extract, flavonoid--Pycnogenol (Pyc), on erythrocyte membrane fluidity was studied. Membrane fluidity was determined using 1-[4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) fluorescence anisotropy. After Pyc action (50 microg/ml to 300 microg/ml), we observed decreases in the anisotropy values of TMA-DPH and DPH in a dose-dependent manner compared with the untreated erythrocyte membranes. Pyc significantly increased the membrane fluidity predominantly at the membrane surface. Further, we observed the protective effect of Pyc against lipid peroxidation, TBARP generation and oxidative hemolysis induced by H2O2. Pyc can reduce the lipid peroxidation and oxidative hemolysis either by quenching free radicals or by chelating metal ions, or by both. The exact mechanism(s) of the positive effect of Pyc is not known. We assume that Pyc efficacy to modify effectively some membrane dependent processes is related not only to the chemical action of Pyc but also to its ability to interact directly with cell membranes and/or penetrate the membrane thus inducing modification of the lipid bilayer and lipid-protein interactions.