Marina V. Zhiryakova

Controllable stability of DNA‐containing polyelectrolyte complexes in water–salt solutions

Destruction of polyelectrolyte complexes (PECs) formed by DNA and synthetic polyamines of different structures was carried out by addition of low molecular weight electrolyte to PEC solution at different pHs. The dissociation was studied by the fluorescence quenching technique using the ability of cationic dye ethidium bromide to intercalate into free sites of DNA double helix followed by ignition of ethidium fluorescence. Structure of amine groups of the polycation was shown to be a decisive factor of PEC stability. PECs formed by polycations with quaternary amine groups, i.e., poly(N-alkyl-4-vinylpyridinium) bromides, poly(N, N-dimethyldiallylammonium) chloride, and ionene bromide, were pH independent and the least tolerant to destruction by the added salt. Primary amine groups of basic polypeptides poly-L-lysine hydrobromide and poly-L-arginine hydrochloride as well as synthetic polycation poly(vinyl-2-aminoethyl ether) provided the best stability of PECs in water-salt solutions under wide pH range. Moderate and pH-dependent stability was revealed for PECs included poly(N,N-dimethylaminoethylmethacrylate) with tertiary amine groups in the chain or branched poly(ethylenimine) with primary, secondary, and tertiary amine groups in the molecule. The data obtained appear to be the basis for design of DNA-containing PECs with given and controllable stability. The design may be accomplished not only by proper choice of polyamine of one or another type, but by using of tailor-made polycations with given composition of amine groups of different structure in the chain as well. Thus, quaternization of a part of tertiary amine groups of poly(N, N-dimethylaminoethylmethacrylate) resulted in expected decrease of stability of DNA-containing PECs in water-salt solutions. The destruction of PEC formed by random copolymer of 4-vinylpyridine and N-ethyl-4-vinylpyridinium bromide was pH sensitive and could be performed under pH and ionic strength closed to the physiological conditions. This result appears to be particularly promising for addressing DNA packed in PEC species to the target cell.

Stability of DNA-containing interpolyelectrolyte complexes in water-salt solutions

Destruction of interpolyelectrolyte complexes (PEC) formed by DNA and different poly(N-alkyl-4-vinylpyridinium) cations was achieved by addition of low molecular weight electrolytes. Monitoring of PEC dissociation was carried out by fluorescence quenching using the ability of the cationic dye ethidium bromide to intercalate into free sites of the DNA double helix accompanied by fluorescence. Degree of polymerization and charge density of the polycations as well as their N-alkyl substituentes (alkyl = methyl, ethyl, and propyl) were shown to be factors influencing the stability of PEC. The ability of added cations and anions to dissociate PEC decreases in the order Ca ++ > Mg ++ >> Li + > Na + > K + >> (CH 3 ) 4 N + and I - > Br > Cl - >> F - which coincides with a decrease of affinity of the same counterions to DNA and to the polycation. The data obtained indicate that the change of the stability of DNA-containing PEC shows the same regularities as revealed for the stability of interpolyelectrolyte complexes formed by oppositely charged flexible synthetic polyelectrolytes in water-salt solutions.

Interaction of Astramol Poly(propyleneimine) Dendrimers with DNA and Poly(methacrylate) Anion in Water and Water–Salt Solutions

Interaction of poly(propyleneimine) dendrimers DAB-dendr-(NH2)x of five generations (x = 4, 8, 16, 32, and 64) with either calf thymus DNA or tagged by pyrenyl groups poly(methacrylate) anion (PMA*) as well as destruction of formed polyelectrolyte complexes by the added sodium chloride were studied by fluorescence quenching techniques. DNA-containing complexes (dendriplexes) were investigated by ethidium bromide assay, whereas formation of PMA* complexes was estimated by fluorescence of the pyrenyl groups that remained free of contact with the dendrimers-quenchers. The ion pairing with DNA phosphate groups was pH-sensitive and accompanied by inaccessibility of a part of the dendrimer amino groups even in slightly acidic media. The growth of the generation number resulted in successive stabilization of the dendriplexes against the added salt. The dendriplexes of all dendrimers except DAB-dendr-(NH2)4 were stable at physiological ionic strength. In contrast to the highly charged cationic polymer poly(N-ethyl-4-vinylpyridinium) bromide of different degrees of polymerization, the dendrimers formed more stable complexes with flexible PMA* rather than with DNA, proving the inaccessibility of a part of the amino groups for the rigid double helix. The revealed regularities appear to be a platform for design of dendriplexes with controllable stability, in particular fulfilling the requirements imposed for gene delivery vehicles.

Competitive reactions in solutions of the complex of chitosan and DNA

The interaction of water-soluble modified chitosan with a polymethacrylate anion and DNA in aqueous and water-salt solutions is studied via the methods of fluorescence quenching with the use of a pyrenyl-labeled polyacid and the intercalated dye ethidium bromide. After passage from neutral to weakly acidic solutions, the stability of the polysaccharide complex with the polymethacrylate anion significantly increases, but, in the case of the DNA-containing complex, stability remains almost invariable. In spite of a much higher stability of complexes against salt in weakly ionized poly(methacrylic acid), the equilibrium of the competitive binding of chitosan shifts toward complexation with DNA, a result that is indicative of the dominant role of electrostatic interactions in complex formation. The capability for sharp and reversible variation in the state of the system in weakly acidic media may become the starting point for development of biotechnological systems for separation of biological mixtures and creation of vectors for delivery of genetic material that are based on cationic biodegradable polysaccharides.

Fluorescence Quenching Technique for Study of Dna-Containing Polyelectrolyte Complexes

Polyelectrolyte complexes (PEC) are the products of cooperative coupling reactions between two unlikely charged polyions of high charge density, in particular with ionogenic groups in each monomer unit of the chain. Of late there has been a widespread interest in research of competitive reactions in PEC’s solutions mimicking some important regulator processes in vivo accompanied by a transfer of charged biopolymers. Data obtained on studying of equilibrium, kinetics and mechanism of the competitive interpolyelectrolyte reactions are summarized in review1. These results lead to crucially new consideration of PEC as macromolecular compounds, permanently exchanging by polyions in water-salt solutions. The ability of PEC to combine high stability with the capacity to take part in the interpolyelectrolyte reactions ensures self- assembly of complex particles in the solutions. Perfect selectivity and high rate of the cooperative interpolyelectrolyte reactions endow PEC with sensitivity to external factors (pH, ionic strength, temperature, etc.) making them self-adjustment systems. Both formation of PEC and their transformation are accomplished by the method of trials and errors via polyions transfer until the equilibrium is achieved.

A water-soluble aromatic dendrimer as a model basis for dual-action drugs

The affinities of two anionic pyrenyl probes for pyridinium high-molecular-mass cations of different topologies—poly(N-ethyl-4-vinylpyridinium bromide) and a water-soluble poly(pyridylphenylene) dendrimer—are studied by the method of fluorescence quenching. The hydrophilic probe carrying three sulfonate groups in a molecule more efficiently interacts with a flexible highly charged linear polycation throughout the studied pH range. The binding of the dendrimer with a relatively hydrophobic probe containing a single carboxyl group is improved by acidification of solutions, and it becomes dominant in weakly acidic solutions. The interaction of DNA with the dendrimer containing the hydrophobic probe has no effect on the formation of the dendriplex and leads to displacement of only a small fraction of the bound probe into solution. Our model studies demonstrate that dual-action dendrimer carriers capable of simultaneous delivery of genetic material and hydrophobic drugs to target cells can be created.

Water-soluble polyelectrolyte complexes of Astramol poly(propyleneimine) dendrimers with poly(methacrylate) anion.

Water-soluble complexes formed by pyrenyl-tagged poly(methacrylate) anion with cationic DAB-dendr-(NH2)x of five generations, x = 4, 8, 16, 32, and 64 were prepared and studied. The ability of the dendrimers to quench the pyrenyl fluorescence was used to monitor formation/dissociation of the complexes by fluorescence quenching technique. In salt-free solutions, dissociation of the complexes occurred in highly acidic and highly alkaline media independently on the dendrimer generation, whereas stability of the complexes against destruction by added salt (NaCl) enhanced markedly with x increase. Phase separations were dependent on pH and charged ratio of the components, but independent of a dendrimer generation. By contrast, in water-salt solutions the generation had a profound impact on phase diagram manifested by a considerable extension of a heterogeneity region as x increased. These findings strongly suggest that the complexes obey the main regularities ascertained for polyelectrolyte complexes of oppositely charged polyions. The revealed possibility of preparing negatively charged and positively charged complexes with controllable stability and solubility demonstrates potentialities of Astramol dendrimers for design self-assembled and self-adjusted systems attractive for biotechnological and biomedical applications.

Cationic nanogels as Trojan carriers for disruption of endosomes

The comparison study of interaction of linear poly(2-dimethyl amino)ethyl methacrylate and its cationic nanogels of various cross-linking with both DNA and sodium poly(styrene sulfonate) has been performed. Although all amino groups of the nanogels proved to be susceptible for protonation, their accessibility for ion pairing with the polyanions was controlled and impaired with the cross-linking. The investigation of nanogels complexes with cells in culture that was accomplished by using of calcein pH-sensitive probe revealed a successive increase in the cytoplasmic fluorescence upon the growth in the cross-linking due to calceine leakage from acidic compartments to cytosol. This regularity implies that amino groups which are buried presumably inside the nanogel are protected against the ion-pairing with polyanions of plasma membrane and hence are able to manifest buffer properties while captured into acidic endosomes, i.e. possess lyso/endosomolytic capacity. These findings suggest that network architecture makes an important contribution to proton sponge properties of weak polycations.