Barbara Klajnert-Maculewicz

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action

This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.

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.

Sugar-modified poly(propylene imine) dendrimers as drug delivery agents for cytarabine to overcome drug resistance.

Maltose-modified poly(propylene imine) glycodendrimers (PPI-m OS) of the 4th generation provide a promising strategy for leukemia treatment. Anticancer therapy with nucleoside analog drugs such as cytarabine (Ara-C) frequently has limited efficacy due to drug resistance, inefficient uptake and accumulation of the drug inside cancer cells where it has to be transformed into the active triphosphate congener. The cationic nature of PPI dendrimers makes it possible to form complexes with nucleotide Ara-C triphosphate forms (Ara-CTP). The aim of this work was to test the concept of applying PPI glycodendrimers as drug delivery devices in order to facilitate the delivery of activated cytarabine to cancer cells to overcome metabolic limitations of the drug. The study has been carried out using 1301 and HL-60 leukemic cell lines as well as peripheral blood mononuclear cells. The results of cytotoxicity and apoptosis assays showed enhanced activity of Ara-C triphosphate form (Ara-CTP) complexed with PPI-m dendrimers in relation to free Ara-C and Ara-CTP against 1301 leukemic cells. Secondly, enhanced uptake and cytotoxicity of Ara-CTP-dendrimers complexes toward 1301 cells with blocked human equilibrative nucleoside transporter - hENT1 suggested that this combination might be a versatile candidate for chemotherapy against resistant acute lymphoblastic leukemia cells with lower expression of hENT1.

Phosphorus dendrimers and photodynamic therapy. Spectroscopic studies on two dendrimer-photosensitizer complexes: Cationic phosphorus dendrimer with rose bengal and anionic phosphorus dendrimer with methylene blue

Dendrimers due to their unique architecture may play an important role in drug delivery systems including chemotherapy, gene therapy and recently, photodynamic therapy as well. We investigated two dendrimer-photosensitizer systems in context of potential use of these systems in photodynamic therapy. The mixtures of an anionic phosphorus dendrimer of the second generation and methylene blue were studied by UV-vis spectroscopy while that of a cationic phosphorus dendrimer (third generation) and rose bengal were investigated by spectrofluorimetric methods. Spectroscopic analysis of these two systems revealed the formation of dendrimer-photosensitizer complexes via electrostatic interactions as well as π stacking. The stoichiometry of the rose bengal-cationic dendrimer complex was estimated to be 7:1 and 9:1 for the methylene blue-anionic dendrimer complex. The results suggest that these polyanionic or polycationic phosphorus dendrimers can be promising candidates as carriers in photodynamic therapy.

PAMAM dendrimer with 4-carbomethoxypyrrolidone—In vitro assessment of neurotoxicity

Cytotoxicity of cationic amino-terminated PAMAM dendrimer and modified PAMAM-pyrrolidone dendrimer was compared. LDH assay and cell visualization technique were employed. Mouse embryonic hippocampal cells (mHippoE-18) were used. The experiments were performed in FBS-deprived medium. Pyrrolidone-modification significantly diminished toxicity of PAMAM dendrimer. The absence of FBS did not reveal significant impact on the toxic effect. Results from LDH assay and MTT test were in good consistency. Low cytotoxicity of PAMAM-pyrrolidone dendrimer increases reliability of the results showing a small impact of this dendrimer on cell viability.

A viologen phosphorus dendritic molecule as a carrier of ATP and Mant-ATP: spectrofluorimetric and NMR studies

In this work, the interactions between a viologen phosphorus dendrimer of generation 0 (VPD) and ATP or 2′-/3′-O-(N′-methylanthraniloyl)-ATP (Mant-ATP) were investigated by NMR and fluorescence spectroscopy methods. ATP and Mant-ATP were used as model molecules of purine and pyrimidine nucleoside analogues (NAs), which are antimetabolites commonly used in anticancer therapy. Complexes of VPD with NAs may help to overcome severe limitations of NAs associated with their low solubility, stability or resistance to cancer cells. The aim of the presented study was to evaluate the stoichiometry and the mechanism of formation of complexes between dendrimers and nucleotides: ATP and Mant-ATP. Moreover, we examined the efficiency of complex formation in relation to temperature, the type of solvent, NaCl concentration, pH and environment polarity. It was observed that viologen phosphorus dendrimers form complexes with ATP and Mant-ATP with high efficiency. Obtained complexes are stabilized by electrostatic and aromatic–aromatic interactions as driving forces.

Cationic phosphorus dendrimers and therapy for Alzheimer's disease

Alzheimers disease (AD) is an age-related neurodegenerative disorder characterized by the aggregation of extracellular β-amyloid and the intracellular microtubule-associated protein Tau. Neurodegeneration is associated inter alia with the activation of microglial cells, neuroinflammation, oxidative stress, and diminished transduction of impulses in cholinergic neurons. Current pharmacotherapy for AD is based mainly on modulation of acetylcholine hydrolysis, administration of non-steroidal anti-inflammatory drugs and antioxidants. Novel drugs with antiamyloidic properties are currently being sought. Cationic phosphorus dendrimers have been proven to modulate amyloidogenesis and stop the aggregation of Tau protein. An ideal drug for AD should demonstrate anti-inflammatory properties, inhibit acetylcholine hydrolysis, and have antioxidant capacity. Cationic phosphorus dendrimers (generation 3 and generation 4) show the foregoing properties. They inhibit acetylcholinesterase activity, can decrease the secretion of TNF-α, and have weak antioxidant effects. The results presented suggest that phosphorus dendrimers may be considered in the future as agents in AD therapy.

In Vitro Studies of Polyhedral Oligo Silsesquioxanes: Evidence for Their Low Cytotoxicity

As scientific literature considers polyhedral oligosilsesquioxanes (POSS) as potential drug delivery systems, it is necessary to check their impact on mammalian cells. Toxicity of octaammonium chloride salt of octaaminopropyl polyhedral oligomeric silsesquioxane (oap-POSS) towards two cell lines: mouse neuroblastoma (N2a) and embryonic mouse hippocampal cells (mHippoE-18) was studied. Experiments consisted of analysis of a cell cycle, cell viability, amount of apoptotic and necrotic cells, and generation of reactive oxygen species (ROS). POSS caused a shift in the cell population from the S and M/G2 phases to the G0/G1 phase. However, the changes affected less than 10% of the cell population and were not accompanied by increased cytotoxicity. POSS did not induce either apoptosis or necrosis and did not generate reactive oxygen species. A cytotoxicity profile of POSS makes it a promising starting material as drug carrier.

Multivalent interacting glycodendrimer to prevent amyloid-peptide fibril formation induced by Cu(II): A multidisciplinary approach

Amyloid peptide fibrillogenesis induced by Cu(II) ions is a key event in the pathogenesis of Alzheimer’s disease. Dendrimers have been found to be active in preventing fibril formation. Therefore, they hold promise for the treatment of Alzheimer’s disease. In this study, the fibrillation mechanism of amyloid peptide Aβ 1-40 was studied by adding Cu(II) in the absence and presence of 4th generation poly(propyleneimine) glycodendrimer functionalized with sulfate groups, using dynamic light scattering (DLS), circular dichroism (CD), fluorescence, electron paramagnetic resonance (EPR) and molecular modeling (MD). The glycodendrimer was non-toxic to mHippoE-18 embryonic mouse hippocampal cells, selected as a nerve cell model, and decreased the toxicity of peptide aggregates formed after the addition of Cu(II). The binary systems of Cu(II)–glycodendrimer, Cu(II)–peptide, and glycodendrimer–peptide were first characterized. At the lowest Cu(II)/glycodendrimer molar ratios, Cu(II) was complexed by the internal-dendrimer nitrogen sites. After saturation of these sites, Cu(II) binding with sulfate groups occurred. Stable Cu(II)–peptide complexes formed within 5 min and were responsible for a transition from an α helix to a β-sheet conformation of Aβ 1-40. Glycodendrimer–peptide interactions provoked the stabilization of the α-helix, as demonstrated in the absence of Cu(II) by the Thioflavin T assay, and in the presence of Cu(II) by CD, EPR, and MD. Formation of fibrils is differentially modulated by glycodendrimer and Cu(II) concentrations for a fixed amount of Aβ 1-40. Therefore, this multidisciplinary study facilitated the recognition of optimal experimental conditions that allow the glycodendrimer to avoid the fibril formation induced by Cu(II).

Fourier transform infrared spectroscopy (FTIR) characterization of the interaction of anti-cancer photosensitizers with dendrimers

The systemic or local administration of a photosensitizer for photodynamic therapy is highly limited by poor selectivity, rapid deactivation and long-lasting skin toxicity due to unfavorable biodistribution. Drug delivery systems based on nanocarriers may help specific and effective delivery of photosensitizers. In the present paper, the interaction of two photosensitizers, methylene blue and rose bengal, with phosphorous cationic and anionic dendrimers as potential nanocarriers, has been characterized. A novel method is presented based on the analysis of the infrared spectra of mixtures of photosensitizer and dendrimer. The capacity of dendrimers to bind the photosensitizers has been evaluated by obtaining the corresponding binding curves. It is shown that methylene blue interacts with both cationic and anionic dendrimers, whereas rose bengal only binds to the cationic ones. Dendrimers are shown to be potential nanocarriers for a specific delivery of both photosensitizers.