Vladimir A. Izumrudov

Polyhistidine-PEG:DNA nanocomposites for gene delivery.

Complexation of plasmid DNA with polycations is a popular method by which to transfer therapeutic nucleic acid sequences to cells. One caveat of the approach is that the positive zeta potential of the complexes facilitates interaction with blood constituents, leading to serum protein adsorption and complement activation. As a countermeasure, investigators have developed polycations combined with polyethylene glycol (PEG) to create complexes with reduced protein adsorption potential. We have designed and synthesized PEG-polyhistidine conjugates to evaluate the material class as potential gene delivery vehicles. Two conjugate architectures (comb-shaped and linear A-B block copolymers) were synthesized and formulated with plasmid DNA. The complexes were characterized with respect to DNA complexation capacity, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection capacity in a model cell line. PEG content of the conjugate significantly influenced the hydrodynamic diameter of the DNA:conjugate composite in aqueous solution. For comb-shaped conjugates steric hindrance attributed to PEG led to a direct relationship between the PEG content and the complex size. Both architectures could condensed plasmid DNA into complexes with hydrodynamic diameters <150 nm. Complexation of DNA with the polyhistidine-PEG conjugates resulted in nanocomposites with negative zeta potentials that retarded DNase I-mediated hydrolysis, and all conjugates showed low cytotoxicity to macrophages cultured in vitro. The transfection efficiency was approximately equivalent to DNA:polylysine complexes. The formulation characteristics and low cytotoxicity suggest that polyhistidine-PEG conjugates may be useful for gene delivery.

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.

Polycomplexes – potential for bioseparation

This review discusses the properties of complexes formed by proteins with polyelectrolytes (PPC) and two polyelectrolyte molecules of opposite charge (PEC). The most highly charged polymers with ionic groups in each monomer unit are considered in this paper. There are all reasons to regard PEC as macromolecular compounds produced as a result of equilibrium reactions with inherent permanent exchange of polyions in water-salt solutions. They combine two properties that might appear at first sight to be mutually exclusive, i.e. rather high stability and lability. Introduction of bioaffinity ligands endows PEC with the recognition capacity sufficient for the purposes of bioseparation and bioanalysis. Antibody-PEC conjugates were successfully used in the immunoassay combining the advantages of both homogeneous and heterogeneous assays and for modeling of chaperone action. The unique properties of polyelectrolyte complexes in combination with bioaffinity ligands makes them promising for the development of highly efficient means of protein isolation, new immunoassay procedures and creation of reversibly soluble biocatalysts.

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.

Structural changes of a protein bound to a polyelectrolyte depend on the hydrophobicity and polymerization degree of the polyelectrolyte

Influence of polyelectrolytes of different chemical structure and degree of polymerization on aggregation and denaturation of the oligomeric enzyme glyceraldehyde-3-phosphate dehydrogenase has been studied to ascertain molecular characteristics of the polymer chains providing the efficient prevention of aggregation of the enzyme without drastic changes in its structure and catalytic activity. The best polymers meeting these requirements were found to be hydrophilic high-molecular-weight polyelectrolytes forming stable complexes with the enzyme. The revealed pronounced negative effect of short polymer chains on the enzyme must be taken into account in the design of protein-polyelectrolyte systems by using thoroughly fractionated polymer samples containing no admixture of charged oligomers.

Preparation and properties of penicillin amidase immobilized in polyelectrolyte complexes

Abstract (1) Immobilization of penicillin amidase (acylamide amidohydrolase, EC 3.5.1.4) from Escherichia coli was carried out on negatively charged particles of the water-soluble polyelectrolyte complex formed by poly(4-vinyl- N -ethylpyridinium bromide) and poly(methacrylic acid) in the ratio 1 : 3. The enzyme was covalently cross-linked to the polycation which was previously modified by cyanuric chloride. (2) Kinetic parameters of benzylpenicillin hydrolysis, catalyzed by immobilized penicillin amidase, were determined and it was shown that with a given method of immobilization, the catalytic efficiency of enzyme action changes slightly. The reaction proceeds in homogeneous aqueous solution without any diffusional difficulties. (3) Deformation of the pH optimum of catalytic activity, an increase of k cat in alkaline media and a sharp increase of the inhibition constant from 2 · 10 −5 to 1 · 10 −3 M caused by the product of the reaction (phenylacetic acid) show a remarkable effect of the negatively charged shell of the polyelectrolyte complex on kinetic parameters of the reaction. (4) Immobilization of the enzyme in polyelectrolyte complex particles leads to the appearance of new properties of the biocatalyst; immobilized penicillin amidase can be reversibly converted to the insoluble state with a slight change in pH and ionic strength of the solution. Transition of the enzyme into the insoluble state results in interruption of the reaction. The conditions for the phase separation of immobilized enzyme in solutions of salts are determined by the composition of polyelectrolyte complex and the nature of the low molecular weight electrolytes. Dissolution of the precipitate leads to quantitative recovery of the initial catalytic activity. (5) The self-regulating enzymatic system was simulated. Control of the activity in the system takes place according to the following scheme: accumulation of product → change in ionic strength of the solution → alteration of microenvironment of the enzyme → decrease in catalytic activity.

Conjugates of monoclonal antibodies with polyelectrolyte complexes - an attempt to make an artificial chaperone

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit muscle is a tetrameric enzyme. Inactivation of GAPDH at 50 degreesC or in the presence of 4 M urea proceeds via formation of inactive dimers followed by their aggregation. Antibodies (clone 6C5) were selected which bind specifically inactive dimers but not native tetramers. The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies were coupled covalently to polyanionic component of the complex, poly(methacrylic) acid. The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium) bromide, resulted in a significant increase in the specific activity of GAPDH. The restoration of specific activity was more complete in the experiments with lower GAPDH concentration and in the samples with lower inactivation degree, conditions where aggregation is less pronounced. Some aggregates are formed at high inactivation degree and high GAPDH concentration and observed as an increase in the solution turbidity. They could be removed by centrifugation. Antibody/polyelectrolyte complex treatment followed by centrifugation to remove insoluble aggregates resulted in nearly complete restoration of enzyme specific activity.

Enzyme-polyelectrolyte noncovalent complexes as catalysts for reactions in binary mixtures of polar organic solvents with water

SummaryThe formation of non-covalent complexes with polyelectrolytes has been suggested to enhance the resistance of enzymes towards inactivation by organic solvents in their homogeneous mixtures with water. Existence of such complexes in water-cosolvent media was proved by experiments with a fluorescence dye, eosin. In the case of catalysis by α-chymotrypsin, formation of the complex with polyelectrolytes produced two major eflects: i) considerable increase in enzyme activity at concentrations of ethanol and N,N-dimethylformamide of 10–30 % v/v; ii) conservation of the enzymatic activity at cosolvent concentrations of more than 40% v/v, where the native enzyme is completely inactive. General character of the observed activation and stabilization phenomena was shown by example of several experimental systems.

Water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride as a nucleic acids vector for cell transfection

To endow the cationic polysaccharides with solubility in the whole pH-range without loss of functionality of the amino groups, different chitosan samples were treated with glycidyltrimethylammonium chloride. Each modified unit of the exhaustively alkylated quaternized chitosan (QCht) contained both quaternary and secondary amino groups. The intercalated dye displacement assay and ζ-potential measurements implied stability of QCht polyplexes at physiological conditions and protonation of the secondary amino groups in slightly acidic media which is favorable for transfection according to proton sponge mechanism. The cytotoxicity and transfection efficacy increased with the chain lengthening. Nevertheless, the longest chains of QCht, 250 kDa were less toxic than PEI for COS-1 cells and revealed comparable and even significantly higher transfection activity of siRNA and plasmid DNA, respectively. Thus, highly polymerized QCht (250 kDa) provided the highest level of the plasmid DNA transfection being 5 and 80 times more active than QCht (100 kDa) and QCht (50 kDa), respectively, and 4-fold more effective than PEI, 25 kDa. The established influence of QCht molecular weight on toxicity and transfection efficacy allows elaborating polysaccharide vectors that possess rational balance of these characteristics.

Interaction of antibodies and antigens conjugated with synthetic polyanions: on the way of creating an artificial chaperone

Recently we have initiated the use of synthetic polyelectrolytes to mimic the action of chaperones in living cells [Dainiak et al., Biochim. Biophys. Acta 1381 (1998) 279-285]. The next step in this direction is done by the synthesis of conjugates of poly(methacrylic acid) (PMAA) with antigen, denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), and with monoclonal antibodies specific for dGAPDH (but not for the native protein). The pH-dependent properties of the conjugates have been studied using turbidimetry and light scattering. The antibody-PMAA and dGAPDH-PMAA conjugates were shown to interact with free dGAPDH and antibodies respectively as well as with each other. Insoluble aggregates of dGAPDH with antibody-PMAA and of antibodies with dGAPDH-PMAA are formed in acidic media. The same situation occurs in the mixture of antibody-PMAA and dGAPDH-PMAA: precipitation takes place in acidic media, whereas soluble associates are formed in neutral solutions. The size of the soluble associates and the number of conjugates in the associate could be regulated by pH. The competition of free dGAPDH and dGAPDH-PMAA for binding with antibody-PMAA and the dynamic release of refolded GAPDH, with no affinity to antibody-PMAA, into solution could be used for simulating chaperone action.