Remnant cationic dendrimers block RNA migration in electrophoresis after monophasic lysis

Cationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) have attractive characteristics for the delivery of nucleic acid and various biomedical applications. Most studies have focused on cationic dendrimer-based intracellular delivery, and very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that PAMAM and PPI dendrimers strongly altered the mobility of RNA in the gels. In addition, the extent of dendrimer-induced alteration in RNA mobility was directly dendrimer-generation-dependent: the alteration was greater with higher-generation dendrimers. We also found that DNA/dendrimer complexes at higher dendrimer to DNA ratios interacted with RNA after isolation while gene expression was maintained. The interactions between RNA and remnant dendrimers after isolation were caused by electrostatic bindings, and we recovered total RNA using high ionic strength solvents (2M NaCl solution) to disrupt the electrostatic forces binding dendrimers to RNA. Because RNA isolation is routinely used for biological applications, such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.     

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon–silicon bonds, but NN16 possesses some oxygen–silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1.By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA–dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation.     

PAMAM dendrimers — diverse biomedical applications. Facts and unresolved questions

In this mini-review a number of novel outcomes, originating from studies in the field of PAMAM dendrimers, are presented and discussed. Owing to the multi-disciplinary nature of dendrimer chemistry it seems important to focus on the relevant topical research of PAMAM dendrimers, including their function, toxicity, surface modifications, and also possible new applications of these spherical polymers. We also consider the possibilities of specific functionalisation of PAMAM dendrimers — both novel ideas and those that have already been reported; as well as their cell-mediated effects (toxic and non-toxic). Then the reactivity of dendrimers’ terminal groups, and their anticipated protective role against modifications of biomacromolecules, are discussed with regard to future developments in biomedical research.     

Parametric-equation representation of biconcave erythrocytes

The representation of the shape of a biconcave erythrocyte by a set of three parametric equations was achieved by using the expressions that transform the curvilinear coordinates from the disc-cyclide coordinate system [denoted J2R; Moon and Spencer (1988), Field Theory Handbook, Springer-Verlag, Berlin] to Cartesian coordinates. The equations are products of elliptic functions, so the challenge was to relate the three major ’shape-defining’ measurements of the human erythrocyte in Cartesian coordinates to three parameters in the new curvilinear coordinates, to give a realistic representation of the shape of the membrane-surface. The relationships between the coefficients of the Cartesian degree-4 surface that describes the discocyte and the coordinate transformation equations were derived with the aid of Mathematica; and the membrane-surface of the cell was drawn using the ParametricPlot3D function in this ‘package’. By having the erythrocyte shape expressed in its new form it is readily amenable to further transformations that might be used to model those changes in shape that are seen when the cells are immersed in media of various osmolalities, or when they change metabolic ’states’. On the other hand, the relationship between the coefficients of the Cartesian expression for the disc-cyclide surface is relevant to image analysis of erythrocytes, as determined by physical methods that rely on Cartesian imaging ’slices’. These methods include confocal microscopy and various nuclear magnetic resonance microimaging procedures.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.     

Synthesis and characterization of nanoscale dendritic RGD clusters for potential applications in tissue engineering and drug delivery

Spatial control over the distribution and the aggregation of arginine-glycine-aspartate (RGD) peptides at the nanoscale significantly affects cell responses. For example, nanoscale clustering of RGD peptides can induce integrins to cluster, thus triggering complete cell signaling. Dendrimers have a unique, highly branched, nearly spherical and symmetrical structure with low polydispersity, nanoscale size, and high functionality. Therefore, dendrimers are a class of ideal scaffold for construction of nanoscale dendritic RGD clusters in which RGD loading degree and cluster size can be finely adjusted. This new type of nanoscale dendritic RGD cluster will aid us to better understand the impact of spatial arrangement of RGD on cellular responses and to engineer RGD to trigger more favorable cellular responses. In this study, nanoscale dendritic RGD clusters were synthesized based on Starburst anionic G3.5 and cationic G4.0 polyamidoamine (PAMAM) dendrimers. The multiple terminal functional groups on the outermost layer of the dendrimer were coupled with RGD tripeptides. Biofunctionalized dendrimer structures were found to be highly dependent on the generation and the extent of peptide modification (ie, number of peptides per PAMAM dendrimer). Fluorescein isothiocyanate (FITC)-conjugated PAMAM dendrimers were utilized to monitor cellular internalization of dendrimers by adherent fibroblasts. Anionic G3.5-based dendritic RGD clusters have been shown to have no negative effect on fibroblast viability and a concentration-dependent effect on lowering cell adhesion on tissue culture polystyrene (TCPS) as that of free RGD. A similar concentration-dependent effect in cell viability and adhesion was also observed for cationic G4.0-based dendritic RGD clusters at lower but not at high concentrations. The results imply that the synthesized nanoscale dendritic RGD clusters have great potential for tissue engineering and drug delivery applications.     

Comet Assay measurements: a perspective

The Comet Assay or single cell gel electrophoresis assay is one of the very widely used assays to microscopically detect DNA damage at the level of a single cell. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). In this assay, the shape, size and amount of DNA within the ‘comet’ play important roles in the determination of the level of damage. The use of a software in particular also provides a range of different parameters, many of which might not be relevant in determining the extent of DNA damage. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of ‘hedgehog’ or ‘clouds’, this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. It is suggested that judicious selection of different parameters, staining methods along with inter-laboratory validation and harmonisation of methodologies will further help in making this assay more robust and widely acceptable for scientific as well as regulatory studies.     

Rethinking cytoskeleton in plant reproduction: toward a biotechnological future?

 Sexual reproduction in plants is intimately connected to the activity of the cytoskeletal apparatus in reproductive cells. Because of the ease with which the pollen tube can be studied, it has become a model for studying many aspects of cell physiology related to the cytoskeleton, such as movement of organelles and vesicles and cell division. However, information about cytoskeletal proteins is still insufficient for determining cytoskeletal functions during reproduction, especially in terms of cell-cell interactions. One reason may be that cytological and biochemical research on the cytoskeleton of pollen and the embryo sac has not been complemented by sufficient research activity at genetic and molecular levels, and few laboratories are currently involved in this work. This might be because of problems in identifying appropriate applied applications of the work that might attract more investigation. Received: 20 January 1999/ Revision accepted: 29 March 1999     

Molecular Determinants of the Cellular Entry of Asymmetric Peptide Dendrimers and Role of Caveolae

Caveolae are flask-shaped plasma membrane subdomains abundant in most cell types that participate in endocytosis. Caveola formation and functions require membrane proteins of the caveolin family, and cytoplasmic proteins of the cavin family. Cationic peptide dendrimers are non-vesicular chemical carriers that can transport pharmacological agents or genetic material across the plasma membrane. We prepared a panel of cationic dendrimers and investigated whether they require caveolae to enter into cells. Cell-based studies were performed using wild type or caveola-deficient i.e. caveolin-1 or PTRF gene-disrupted cells. There was a statistically significant difference in entry of cationic dendrimers between wild type and caveola-deficient cells. We further unveiled differences between dendrimers with varying charge density and head groups. Our results show, using a molecular approach, that (i) expression of caveola-forming proteins promotes cellular entry of cationic dendrimers and (ii) dendrimer structure can be modified to promote endocytosis in caveola-forming cells.     

Genotoxicity of poly(propylene imine) dendrimers

Dendrimers are highly branched macromolecules with the potential in biomedical applications. Due to positively charged surfaces, several dendrimers reveal toxicity. Coating peripheral cationic groups with carbohydrate residues can reduce it. In this study, the cytotoxicity and genotoxicity of three types of 4th generation poly(propylene imine) dendrimers were investigated. Peripheral blood mononuclear cells (PBMCs) were treated with uncoated (PPI-g4) dendrimers, and dendrimers in which approximately 40% or 90% of peripheral amino groups were coated with maltotriose (PPI-g4-OS or PPI-g4-DS) at concentration of 0.05, 0.5, 5 mg/ml. Abbreviations OS and DS stand for open shell and dense shell respectively, that describes the structure of carbohydrate modified dendrimers. After 1 h of cell incubation at 37°C, the MTT and comet assays were performed. PPI dendrimers demonstrated surface-modification-degree dependent toxicity, although genotoxicity of PPI-g4 and PPI-g4-OS measured by the comet assay was concentration dependent up to 0.5 mg/ml and at 5 mg/ml the amount of DNA that left comet's head decreased. Results may suggest a strong interaction between dendrimers and DNA, and furthermore, that coating PPI dendrimers by maltoriose is an efficient method to reduce their genotoxicity what opens the possibilities to use them as therapeutic agents or drug carriers.     

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

Antimicrobial peptides (AMPs), with their extraordinary properties, such as broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new antibiotics. Despite their various mechanisms of action, the interaction of AMPs with the bacterial cell membrane is the key step for their mode of action. Moreover, it is generally accepted that the membrane is the primary target of most AMPs, and the interaction between AMPs and eukaryotic cell membranes (causing toxicity to host cells) limits their clinical application. Therefore, researchers are engaged in reforming or de novo designing AMPs as a ‘single-edged sword’ that contains high antimicrobial activity yet low cytotoxicity against eukaryotic cells. To improve the antimicrobial activity of AMPs, the relationship between the structure and function of AMPs has been rigorously pursued. In this review, we focus on the current knowledge of α-helical cationic antimicrobial peptides, one of the most common types of AMPs in nature.     

The interaction mechanism between lipopeptide (daptomycin) and polyamidoamine (PAMAM) dendrimers

The interaction mechanism of lipopeptide antibiotic daptomycin and polyamidoamine (PAMAM) dendrimers was studied using fluorescence spectroscopy. The fluorescence changes observed are associated with daptomycin–dendrimer interactions. The binding isotherms were constructed by plotting the fluorescence difference at 460 nm from kynurenine (Kyn‐13) of daptomycin in the presence and absence of dendrimer. A one‐site and two‐site binding model were quantitatively generated to estimate binding capacity and affinity constants from the isotherms. The shape of the binding isotherm and the dependence of the estimated capacity constants on dendrimer sizes and solvent pH values provide meaningful insight into the mechanism of interactions. A one‐site binding model adequately describes the binding isotherm obtained under a variety of experimental conditions with dendrimers of various sizes in the optimal binding pH region 3.5 to 4.5. Comparing the pH‐dependent binding capacity with the ionization profiles of daptomycin and dendrimer, the ionized aspartic acid residue (Asp‐9) of daptomycin primarily interact with PAMAM cationic surface amine. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The influence of cationic dendrimers on antibacterial activity of phage endolysin against P. aeruginosa cells

Nowadays, the researchers make a big effort to find new alternatives to overcome bacterial drug resistance. One option is the application of bacteriophage endolysins enable to degrade peptidoglycan (PG) what in consequence leads to bacterial cell lysis. In this study we examine phage KP27 endolysin mixed with poly(propyleneimine) dendrimers to evaluate an antimicrobial effect against Pseudomonas aeruginosa. Polycationic compounds destabilize bacterial outer membrane (OM) helping endolysins to gain access to PG. We found out that not only bacterial lipopolysaccharide (LPS) is the main hindrance for highly charged cationic dendrimers to disrupt OM and make endolysin reaching the target but also the dendrimer surface modification. The reduction of a positive charge and concentration in maltose poly(propyleneimine) dendrimers significantly increased an antibacterial effect of endolysin. The application of recombinant lysins against Gram-negative bacteria is one of the future therapy options, thus OM permeabilizers such as cationic dendrimers may be of high interest to be combined with PG-degrading enzymes.     

Interactions between PAMAM dendrimers and bovine serum albumin

Dendrimers are a new class of polymeric materials. They are globular, highly branched, monodisperse macromolecules. Due to their structure, dendrimers promise to be new, effective biomedical materials as oligonucleotide transfection agents and drug carriers. More information about biological properties of dendrimers is crucial for further investigation of dendrimers in therapeutic applications.In this study the mechanism of interactions between polyamidoamine (PAMAM) dendrimers and bovine serum albumin (BSA) was examined. PAMAM dendrimers are based on an ethylenediamine core and branched units are constructed from both methyl acrylate and ethylenediamine. We used three types of PAMAM dendrimers with different surface groups (-COOH, -NH(2), -OH). As BSA contains two tryptophan residues we were able to evaluate dendrimers influence on protein molecular conformation by measuring the changes in the fluorescence of BSA in the presence of dendrimers. Additionally experiments with a fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS) were carried out. The differential scanning calorimetry (DSC) was chosen to investigate impact on protein thermal stability upon the dendrimers.Our experiments showed that the extent of the interactions between BSA and dendrimers strongly depends on their surface groups and is the biggest for amino-terminated dendrimers.     

Peptide and glycopeptide dendrimers and analogous dendrimeric structures and their biomedical applications

The size of information that can be stored in nucleic acids, proteins, and carbohydrates was calculated. The number of hexamers for peptides is 64,000,000 (20⁶) and seems to be impressive in comparison with 4,096 (4⁶) hexanucleotides, but the number of isomers of hexasaccharides is 1.44 × 10¹⁵. Carbohydrates are therefore the best high-density coding system. This language has been named glycocode resp. sugar code. In comparison with peptide dendrimers, the amount of information carried by glycopeptide dendrimers or glycodendrimers is therefore much higher. This is reflected by the variability of structures and functions (activities). This review is about the broad area of peptide and glycopeptide dendrimers. The dendrimeric state and physicochemical properties and general consequences are described, together with a cluster effect. The impact of cluster effect to biological, chemical, and physical properties is discussed. Synthesis of dendrimers by convergent and divergent approaches, “Lego” chemistry, ligation strategies, and click chemistry is given with many examples. Purification and characterization of dendrimers by chromatographic methods, electromigration methods, and mass spectrometry are briefly mentioned. Different types of dendrimers with cyclic core, i.e. RAFTs, TASPs and analogous cyclic structures, carbopeptides, carboproteins, octopus glycosides, inositol-based dendrimers, cyclodextrins, calix[4]arenes, resorcarenes, cavitands, and porphyrins are given. Dendrimers can be used for creation of libraries, catalysts, and solubilizing agents. Biocompatibility and toxicity of dendrimers is discussed, as well as their applications in nanoscience, nanotechnology, drug delivery, and gene delivery. Carbohydrate interactions of glycopeptide dendrimers (bacteria, viruses, and cancer) are described. Examples of dendrimers as anti-prion agents are given. Dendrimers represent a fast developing area which partly overlaps with nanoparticles and nanotechnologies.     

The breakdown of bilayer lipid membranes by dendrimers

The BLM-system for studying the electrophysical properties of bilayer lipid membranes (BLM) was applied to investigate interactions between polyamidoamine (PAMAM) dendrimers and lipid bilayers. The cationic PAMAM G5 dendrimer effectively disrupted planar phosphatidylcholine membranes, while the hydroxyl PAMAM-OH G5 and carboxyl PAMAM G4.5 dendrimers had no significant effect on them.     

Use of cyclodextrin and its derivatives for increased transformation efficiency of competent bacterial cells

Methodologies for introduction of DNA into cells are essential in molecular genetics and vital for applications such as genetic engineering and gene therapy. The use of cyclodextrins (CyDs) for increased efficiency of introducing DNA into eukaryotic cells (transfection) has been reported, but CyDs’ effect on the introduction of DNA into bacterial cells (transformation) is unknown. Here, we have investigated the potential of using CyDs in the transformation of chemically competent in-house, commercially available, and, on non-competent bacterial cells, with plasmid DNA of two different sizes. Possible interactions between CyDs and DNA were studied with nuclear magnetic resonance (NMR) spectroscopy. The presence of CyDs resulted in an up to fourfold increment of the transformation rate for in-house cells, with β-CyD and derivates giving the strongest effect. For commercial cells and transformation with megaplasmids, a more moderate effect around 1.4-fold was obtained. However, CyDs have little or no effect on DNA uptake by noncompetent cells. Results obtained from NMR spectroscopy show no interactions between CyDs and DNA-like molecules, which indicated that the CyDs’ effect is related to the bacterial cell wall. An erratum to this article can be found at     

Self-sterility in the mutant ‘Zigui shatian’ pummelo (<Emphasis Type="Italic">Citrus grandis</Emphasis> Osbeck) is due to abnormal post-zygotic embryo development and not self-incompatibility

‘Shatian’ pummelo (Citrus grandis Osbeck), one of the main citrus cultivars in China, is self-incompatible, and its pollen tubes are believed to be arrested in style after self-pollination.We have characterized one ‘Shatian’ pummelo mutant, named ‘Zigui shatian’ pummelo. The mutant pummelo had identical DNA ploidy level, morphology (leaf shape, stoma size and density, pollen shape and size) and developmental progress of pistil and male organs to that of the common ‘Shatian’ pummelo. However, unlike the common ‘Shatian’ pummelo, ‘Zigui shatian’ is self-compatible since its pollen tubes can self-pollinate allowing for successful fertilization. Histological analyses of ‘Shatian’ pummelo further verified abnormal post-zygotic development which led to seed abortion. Simple sequence repeats (SSR) analysis revealed polymorphism in 1 of the 120 primers screened showing that ‘Zigui shatian’ and ‘Shatian’ pummelo are different at the DNA level. Taken together, these data suggested mutant ‘Zigui shatian’ pummelo might be derived from ‘Shatian’ pummelo with self-sterility by self-incongruity after self-fertilization.