Katarzyna Milowska

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).

PAMAM G4 dendrimers affect the aggregation of α-synuclein.

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinsons 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.

Viologen-Phosphorus Dendrimers Inhibit α-Synuclein Fibrillation

Inhibition of α-synuclein (ASN) fibril formation is a potential therapeutic strategy in Parkinsons 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.

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 Parkinsons 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.

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 peoples life.

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (A). Mechanisms of interaction.

This paper examines a perspective on the use of newly engineered nanomaterials as effective and safe carriers of genes for the therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus and carbosilane) were complexed with anticancer siRNA and their biophysical properties of the dendriplexes analyzed. The potential of the dendrimers as nanocarriers for anticancer siBcl-xl, siBcl-2, siMcl-1 siRNAs and a siScrambled sequence was explored. Dendrimer/siRNA complexes were characterized by methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. Some of the experiments were done with heparin to check if siRNA can be easily disassociated from the complexes, and whether released siRNA maintains its structure after interaction with the dendrimer. The results indicate that siRNAs form complexes with all the dendrimers tested. Oligoribonucleotide duplexes can be released from dendriplexes after heparin treatment and the structure of siRNA is maintained in the case of PAMAM or carbosilane dendrimers. The dendrimers were also effective in protecting siRNA from RNase A activity. The selection of the best siRNA carrier will be made based on cell culture studies (Part B).

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.

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.

Dendrimers complexed with HIV-1 peptides interact with liposomes and lipid monolayers.

AIMS We have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD). METHODS Interaction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir-Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy. RESULTS All dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive. CONCLUSIONS HIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.

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.