Structural polymorphism of liquid-crystalline dispersions of double-stranded DNA complexed with synthetic polycations

Linear double-stranded DNA molecules interact with positively charged polyconidine molecules in aqueous salt solutions to yield liquid-crystalline dispersions (LCDs) with a mean particle diameter of ～6000 Å. The packing density of (DNA-polycation) complexes differs among LCD particles formed at different ionic strengths. X-ray data on the liquid-crystalline phases of (DNA-polyconidine) complexes formed under different conditions were compared with a phase diagram, reflecting polymorphism of liquid crystals of linear double-stranded DNA. It was shown that LCD was hexagonal at 0.15 M ≤ C _NaCl < 0.4 M and cholesteric at 0.4 M ≤ C _NaCl < 0.55 M. Cholesteric LCD displayed abnormal optical activity in the circular dichroism spectrum. A similar situation was observed with poly(2,5-ionene), another polycation differing in chemical structure from polyconidine. The results demonstrated structural polymorphism of (DNA-polycation) LCDs. It was assumed that the packing mode of (DNA-polycation) complexes in LCD particles can be regulated by changing NaCl concentration. The mechanism generating the cholesteric liquid-crystalline state of DNA in a narrow range of NaCl concentrations is discussed.     

Temperature-induced changes of the packing of double-stranded linear DNA molecules in particles of liquid-crystalline dispersions

The circular dichroism spectra of liquid-crystalline dispersions obtained by phase exclusion of linear double-stranded DNA molecules from aqueous saline solutions of polyethylene glycol (120 ≤ C_PEG ≤ 300 mg/mL) have been investigated. The formation of liquid-crystalline dispersions at polyethylene glycol concentrations ranging from 120 to 200 mg/mL was accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism; this is indicative of cholesteric packing of the double stranded DNA molecules in the particles of the dispersion. Liquid-crystalline dispersions formed at PEG concentrations higher than 220 mg/mL and room temperature did not show any abnormal bands in the circular dichroism spectra; this is indicative of hexagonal packing of double-stranded DNA molecules in the particles of the dispersions. Heating of optically inactive liquid crystal dispersions induced a transition of the dispersions into a different state accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism. This transition is considered within the concept of the transformation of a hexagonal packing of DNA molecules into a cholesteric packing. A qualitative mechanism of such a transition is proposed that is formulated in the terms of the “quasinematic” layers of double-stranded DNA molecules that change their spatial orientation under the competing influences of the osmotic pressure of the solvent, orientational elasticity of the cholesteric packing, and thermal fluctuations.     

Formation of Liquid-Crystalline Dispersions of Double-Stranded DNA–Chitosan Complexes

Right-handed helical double-stranded DNA molecules were shown to interact with chitosans to form under certain conditions (chitosan molecular weight, content of amino groups, distance between amino groups, ionic strength and pH of solution) cholesteric liquid-crystalline dispersions characterized by abnormal positive band in CD spectrum in the absorption region of DNA nitrogen bases. Conditions were found for the appearance of intense negative band in CD spectrum upon dispersion formation. In some cases, no intense band appeared in CD spectrum in spite of dispersion formation. These results indicate not only the multiple forms of liquid-crystalline dispersions of DNA–chitosan complexes but also a possibility to control the spatial properties of these complexes. The multiplicity of liquid-crystalline forms of DNA–chitosan complexes was attempted to explain by the effect of character of dipoles distribution over the surface of DNA molecules on the sense of spatial twist of cholesteric liquid crystals resulting from molecules of the complexes.     

DNA complexes with polycations useful for delivery of DNA into cells

Generation and physicochemical properties of complexes formed by high-molecular thymus DNA and plasmid DNA with synthetic polymers of (dimethyl amino)ethyl methacrylate, (diethyl amino)ethyl methacrylate, and poly(vinyl amine) were studied in solutions of different ionic strength using low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The complexes were tested for toxicity with T98G cell cultures. Condensation of DNA was shown to occur when the ratio of charged groups in the polycations and DNA exceeded unity. This condensation manifested itself as an increase in the optical density of DNA solutions. Condensation-associated changes in the dimensions of DNA molecules were determined, and phase diagrams of DNA-polycation systems were analyzed in the presence of NaCl. MTT analysis revealed no toxicity of these complexes.     

Serum albumins have five sites for binding of cationic dendrimers

The detailed analysis of the interaction between PAMAM G4 dendrimer and serum albumins was performed using circular dichroism, isothermal titration calorimetry, capillary electrophoresis, zeta-potential and fluorescence polarization. It was shown that serum albumins and PAMAM G4 dendrimer form the complex with stoichiometry of 4-6:1 for G4:HSA and 4-5:1 for G4:BSA molar ratio. The possible sites of PAMAM G4 dendrimers binding to protein surface were discussed. Also, it has been proposed that dendrimer does not significantly affect the protein secondary structure studied by circular dichroism.     

Formation of liquid-crystalline dispersions of double-stranded DNA-chitosan complexes

Right-handed helical double-stranded DNA molecules were shown to interact with chitosans to form under certain conditions (chitosan molecular weight, content of amino groups, distance between amino groups, ionic strength and pH of solution) cholesteric liquid-crystalline dispersions characterized by abnormal positive band in CD spectrum in the absorption region of DNA nitrogen bases. Conditions were found for the appearance of intense negative band in CD spectrum upon dispersion formation. In some cases, no intense band appeared in CD spectrum in spite of dispersion formation. These results indicate not only the multiple forms of liquid-crystalline dispersions of DNA-chitosan complexes but also a possibility to control the spatial properties of these complexes. The multiplicity of liquid-crystalline forms of DNA-chitosan complexes was attempted to explain by the effect of character of dipoles distribution over the surface of DNA molecules on the sense of spatial twist of cholesteric liquid crystals resulting from molecules of the complexes.     

Nanogel formation consisting of DNA and poly(amido amine) dendrimer studied by static light scattering and atomic force microscopy

Binding of a poly(amido amine) dendrimer with salmon testes DNA in an aqueous solution of 0.01 M NaCl at pH 6.5 has been investigated. It was shown from the physicochemical experiments of static light scattering and ultra-violet and circular dichroism spectroscopy that at a 0.2 mixing ratio of dendrimer/DNA (number ratio of NH2 groups in dendrimer vs phosphate groups in DNA) significant conformational change of the DNA occurred owing to the binding of dendrimers on the DNA chain. The dendrimer/DNA complexes formed aggregates (nanogels) when the mixing ratio was increased above 0.2. Weight-averaged molecular weight, radius of gyration, and turbidity measurements revealed that the size of the aggregates increased up to a mixing ratio of 0.8. Atomic force microscopic images certified the formation of complexes and the morphology of the nanogels.     

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 Parkinson's 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.     

Structural polymorphism of the liquid-crystalline dispersions formed from the double-stranded DNA molecules complexed with synthetic polycations

The double-stranded, linear DNA molecules form the liquid-crystalline dispersions (LCD) in water-salt solutions containing positively charged polyconidin molecules. It was established from the analysis of the absorption spectra of the LCDs formed from (DNA-polyconidin) complexes, that the mean size of the particles of these dispersions is equal to -6000 angstroms. The small-angle X-ray data show, that in the LCD particles different density of packing of the (DNA-polycation) complexes is realized. The comparison of the X-ray data of the liquid-crystalline phases of (DNA-polyconidin) complexes formed under various conditions with the phase diagram, that reflects the polymorphism of the linear double-stranded DNA liquid crystals, demonstrates that the hexagonal mode of the LCD packing is existing in 0.15-0.4 M NaCl solutions, whereas in 0.4-0.55 M NaCl solutions-- the cholesteric one. As a result of specific spatial organization the cholesteric LCD possesses of an abnormal optical activity in the CD spectrum. The similar situation takes place in the case of another synthetic polycation--poly(2,5-ionen), whose chemical structure differs from that of polyconidin. Thus, the structural polymorphism of the (DNA-polyconidine) LCDs was evidenced. It means that change of NaCl concentration opens a gate to control the spatial packing of the molecules of (DNA-polycation) complexes in the particles of LCDs. The supposition about mechanism of formation of the DNA cholesteric liquid-crystalline state in the narrow interval of NaCl concentrations was suggested.     

The formation of liquid-crystalline dispersions of DNA-chitosan complexes under the conditions of molecular crowding

The formation of liquid-crystalline dispersions from DNA-chitosan complexes in polyethyleneglycol-containing solutions was studied. It was shown that the molecular crowding affects neither the efficiency of binding of chitosan molecules to DNA nor the mode of spatial packing of DNA-chitosan complexes in particles of liquid-crystalline dispersions.     

A stopped-flow kinetic study of the assembly of nonviral gene delivery complexes

Stopped-flow circular dichroism and fluorescence spectroscopy are used to characterize the assembly of complexes consisting of plasmid DNA bound to the cationic lipids dimethyldioctadecylammonium bromide and 1, 2-dioleoyl- 3-trimethylammonium-propane and a series of polyamidoamine dendrimers. The kinetics of complexation determined from the stopped-flow circular dichroism measurements suggests complexation occurs within 50 ms. Further analysis, however, was precluded by the presence of mixing (shear) artifacts. Stopped-flow fluorescence employing the high-affinity DNA dyes Hoechst 33258 and YOYO-1 was able to resolve two sequential steps in the assembly of complexes that are assigned to binding/dehydration and condensation events. The rates of each process were determined over the temperature range of 10-50 degrees C and activation energies were determined from the slope of Arrhenius plots. The behavior of polyamidoamine dendrimers can be separated into two classes based on their differing binding modes: generation 2 and the larger generations (G4, G7, and G9). The larger generations have activation energies for binding that follow the trend G4 > G7 > G9. The activation energies for condensation (compaction) of complexes composed of these same dendrimers have the opposite trend G9 > G7 > G4. It is postulated that a balance between a more energetically favorable condensation and less favorable binding may prove beneficial in enhancing gene delivery.     

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Lysine dendrimers as vectors for delivering genetic constructs to eukaryotic cells

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for delivering DNA to eukaryotic cells and provide a basis for constructing more refined nonvirus module carriers.     

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Complex formation between endogenous toxin bilirubin and polyamidoamine dendrimers: a spectroscopic study

The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442-455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 degrees C: for PAMAM G4 dendrimer, (2.4+/-1.4) x 10(6) (mol/l)(-1) and 0.07+/-0.012; for PAMAM G4-OH dendrimer, (3.1+/-1.3) x 10(6) (mol/l)(-1) and 0.08+/-0.014; for PAMAM G5 dendrimer, (7.6+/-3.6) x 10(6) (mol/l)(-1) and 0.09+/-0.02; and for PAMAM G5-OH dendrimer, (8.5+/-3.2) x 10(6) (mol/l)(-1) and 0.09+/-0.02. These effects can be explained by the formation of bilirubin-PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins.     

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.     

Lysine dendrimers as vectors for delivery of genetic constructs to eukaryotic cells

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for DNA delivery to eukaryotic cells and provide a basis for constructing more refined nonviral module carriers.     

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.     

DNA complexes with polycations used for delivery of DNA into cells

The formation and physicochemical properties of high-molecular thymus and plasmid DNA complexes with synthetic polymers based on (dimethyl-amino)ethyl methacrylate (DMAEM), (diethyl-amino)ethyl methacrylate (DEAEM), and polyvinyl amine (PVA) were investigated in solutions of different ionic strength by low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The toxicity of complexes in T98G cells was studied. It was shown that, when the ratio of polycations to DNA charged groups concentration (N+/P) reaches values > 1, DNA condensation occurs. It is accompanied by increasing optical density of solutions. Changes in DNA size after condensation were estimated. Phase diagrams of systems DNA/polycation in the presence of NaCl were obtained. It was shown by MTT-analysis that DNA complexes with polycations in the range of concentrations used have low toxicity.     

SAXS-data-based structural modeling of DNA–gadolinium complexes fixed in particles of cholesteric liquid-crystalline dispersions

Structure of cholesteric liquid-crystalline dispersions (CLCDs) formed by double-stranded DNA molecules and treated with gadolinium salts was studied by small-angle X-ray scattering (SAXS). The obtained SAXS data open the way for structural modeling of these complexes to obtain a reasonable explanation for the correlated decrease in amplitude of an abnormal negative band in the circular dichroism (CD) spectra and the characteristic Bragg peak in the experimental small-angle X-ray scattering curves observed on treatment of CLCD by gadolinium salts. Model simulations of different kinds of structural organizations of the DNA–gadolinium complex were performed using novel SAXS data analysis methods in combination with several new, complementary modeling techniques, enabling us to build low-resolution three-dimensional structural models of DNA–gadolinium complexes fixed in CLCD particles. The obtained models allow us to suggest that a change takes place in the helical twist of quasinematic layers formed by these molecules at high concentrations of gadolinium salt. This change in the twist can be used to explain the experimentally observed increase in amplitude of an abnormal band in the CD spectra of DNA CLCD.