Biophysical characterization of glycodendrimers as nano-carriers for HIV peptides.

This paper examines the formation and stability of nano-complexes that could provide a new therapeutic approach against HIV-1 infection. Poly(propylene imine) glycodendrimers decorated with 2(nd) generation cationic carbosilane dendrons were generated and their use in polyplex formation checked. Owing to their positively-charged terminal amino groups the hybrid glycodendrimers can bind anionic peptides. It was shown that they form nano-complexes with the HIV-derived peptides P24, Gp160 and Nef. Complexes 130-190 nm in size were formed in molar ratios (dendrimer/ peptide) of (3-4):1. These were sufficiently stable over time and at different pHs. The results obtained suggest that the hybrid dendrimers studied can be considered as alternative carriers for delivering HIV peptides to dendritic cells.

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.

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.

Non-viral engineering of skin precursor-derived Schwann cells for enhanced NT-3 production in adherent and microcarrier culture.

Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.

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.

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.

Fourth generation phosphorus-containing dendrimers: prospective drug and gene delivery carrier.

Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate.

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.

Does fluorescence of ANS reflect its binding to PAMAM dendrimer?

The analysis of binding between cationic PAMAM G5 dendrimer and anionic fluorescent probe using fluorescence and equilibrium dialysis has been made. It was found that at low concentrations of ANS the double fluorimetric titration technique can be successfully used for quantitative analysis of binding of ANS to dendrimer. Based on fluorescence and dialysis data the constants of binding and the number of binding centers were calculated for binding of ANS to PAMAM G5 dendrimer: K(b) is approx. (0.5-1)x10(5)M(-1) and n is (0.5-0.7).

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.

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.

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.