V.A. Kabanov

From synthetic polyelectrolytes to polymer-subunit vaccines

The results of many years of collaborative research by chemists and immunologists in the area of application of synthetic polyelectrolytes in immunology are reviewed. Linear synthetic polyelectrolytes with diverse structures (which are not structural analogs of biopolymers and, hence, not antigenic because they are unknown to the immune system), when introduced into the organism, noticeably intensify the formation, migration, and dissemination of stem cells, which are precursors of all specialized cells including functional immune cells. In addition, synthetic polyelectrolytes, when introduced in mixtures with typical antigens (proteins, natural microbial polysaccharides, and their synthetic analogs), serve as immunostimulants enhancing immune response by several times. Moreover, individual bacterial or viral antigens, not sufficiently active by themselves, induce specific immune response enhanced by orders of magnitude if chemically bound to synthetic polyelectrolytes. Such conjugates being preliminarily administrated, protect organisms from absolutely mortal doses of the corresponding bacteria or viruses. The nontoxic immunostimulant was developed: the ternary copolymer of 1,4-ethylenepiperazine,1,4-ethylenepiperazine-N-oxide, and (N -carboxymethylene)-1,4-ethylenepiperazinium bromide (brand name “polyoxidonium ”™) permitted for human administration. The conjugate of polyoxidonium with hemagglutinin and neuraminidase, protein subunits of influenza viruses, has appeared as the first non-Pasteurian vaccine, which now is successfully used in Russia (about 50 million immunized people for the last 7 years). The physicochemical mechanisms of the biological effect of these compounds and challenges of the further use of the approach developed are considered in the review.

Solvation effects and reactivity of free pyridine residues in macromolecules of poly-4-vinylpyridine derivatives

Abstract Water-soluble derivatives of poly-4-vinylpyridine (PVP) were prepared by means of quaternization of PVP with ethyl bromide, butyl bromide, hexyl-bromide and bromacetic acid. In addition, hydrolysis of p-nitrophenylacetate (NPA) and 3-nitro-4-acetoxybenzoic acid (NABA) catalyzed by these polymers and 4-ethylpyridine as analogue were studied spectrophotometrically both in water and alcohol-water mixtures at 25° and pH 7·8. The second-order rate of NPA hydrolysis (Kt) was independent of the length of alkyl groups and the salt concentration. While an increase of the degree of quaternization (β) for PVP derivatives resulted in a reduction of the value of Ki in water, in 37–50 vol per cent alcohol-water there was the opposite effect. At large values of β, Ki did not depend on the content of ethanol. The bell-shaped dependence of Ki on β was shown in the case of amionic NABA. The value of Ki proved to be very sensitive to both the nature of salt and the salt concentration in aqueous solution. The use of viscosity, turbidimetry and potentiometry for studying these polymers allowed the role of the quaternized pyridine residue to be elucidated. The peculiarities of the kinetic behaviour of the free basic group were explained by the hydrophilic nature of the quaternized PVP changing as the degree of quaternization is increased so causing an exclusion of the substrate molecules from the polyion.

MODULATION OF INTERACTION OF POLYCATIONS WITH NEGATIVE UNILAMELLAR LIPID VESICLES

Interactions of small unilamellar negative vesicles composed of diphosphatidylglycerol (cardiolipin, CL2−), 20 mol%, and phosphatidylcholine (egg yolk lecithin, EL), 80 mol%, with various cationic polymers (CP) derived from poly(4-vinylpyridine) (PVP) were studied in water and water–salt solutions by means of photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. The linear charge density and hydrophilic lipophilic balance of CPs were varied by quaternization of PVP with various amounts of different alkyl bromides (ethyl-(2), heptyl-(7), dodecyl-(12), cetyl-(16)). Substantial differences were observed in the behavior of exhaustingly N-ethylated PVP (CP2) and PVP N-ethylated to 50 mol% (CP2(50)) or 30 mol% (CP2(30)). All of them adsorb to the CL2−/EL vesicle membrane, neutralizing the surface negative charge and causing aggregation of the vesicles. However, CP2, a polycation with a maximum linear charge density, strongly enhances transfer of the negative lipid ions from the inner to outer bilayer leaflet, while CP2(50) and CP2(30) do not. Adsorbed CP2 does not disturb integrity of the vesicle membrane and can be completely removed from the surface of aggregated vesicles by adding a simple salt (NaCl) or a negative linear polyelectrolyte (polyacrylic acid (PAA) sodium salt). Such removal is followed by release of the original vesicles. In contrast to that, adsorbed CP2(50) or CP2(30) produce some leak through the lipid bilayer and cannot be completely desorbed either by increasing ionic strength or adding an excess of PAA. The probable reason of these differences is discussed. PVP partially N-alkylated with dodecyl or cetyl bromides (3 mol%) and then completely N-ethylated (CP2,12 and CP2,16), also having a maximum linear charge density, adsorbs to the negative vesicle surface as a result of both electrostatic binding and hydrophobic interaction. Bulky hydrocarbon pendant groups incorporate into the inner bilayer compartment. Similarly to CP2(50) and CP2(30), CP2,12 and CP2,16 cannot be removed from the surface either by adding the simple salt, or an excess of PAA. However, in contrast to CP2(50) and CP2(30), the polycations with the bulky hydrocarbon pendant groups do not cause any leak through the vesicle membrane. Finally, we have succeeded to prepare the ternary vesicles also composed of 20 mol% of CL2−, but partially replacing EL for polyoxyethylene 20 cetyl ether (Brij 58) (up to 30 mol%). The CL2−/EL/Brij vesicle carries a hydrophilic corona formed by polyoxyethylene chains exposed into water, while hydrophobic cetyl radicals are incorporated in the lipid bilayer. The CL2−/EL/Brij vesicles adsorb all studied CPs similar to the binary CL2−/EL vesicles. This means that polyoxyethylene corona is permeable for polycationic species restricting neither electrostatic binding nor incorporation of bulky hydrocarbon groups of CP2,16 into the membrane. However, the corona effectively stabilizes the CP-vesicle complexes against aggregation when the membrane surface is neutralized.

Reversibility of structural rearrangements in the negative vesicular membrane upon electrostatic adsorption/desorption of the polycation.

Interaction of small unilamellar vesicles (SUVs), composed of negative diphosphatidylglycerol (cardiolipin, CL(2-)) and neutral dipalmitoylphosphatidylcholine (DPPC), with poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) was studied in water solution above and below the vesicular membrane melting point by means of differential scanning calorimetry, photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. It has been found that CL(2-) species are homogeneously distributed within DPPC-CL(2-) SUV membrane leaflets and between them. Interaction of PEVP with DPPC-CL(2-) SUVs led to drastic structural rearrangements in the membrane if it was in the fluid state (liquid SUVs). Negative CL(2-) molecules migrated from the inner to the outer membrane leaflet and segregated in the vicinity of adsorbed PEVP chains. In addition, PEVP adsorption terminated completely the exchange of lipid molecules between the SUVs. At the same time, the integrity of liquid SUVs contacting PEVP remained unchanged. Since the interaction of PEVP with liquid SUVs was predominantly electrostatic in nature, the polycation could be completely removed from the vesicular membrane by addition of an excess of polyacrylic acid (PAA) polyanions forming a more stable electrostatic complex with PEVP. Removal of PEVP resulted in complete resumption of the original distribution of lipids in lateral and transmembrane directions as well as intervesicular lipid exchange. In contrast, PEVP interacting with DPPC-CL(2-) SUVs formed defects in the vesicular membrane if it was in the gel state (solid SUVs). Such interaction was contributed not only by electrostatic but most likely by hydrophobic interactions involving the defected membrane sites. PEVP kept contacting solid SUVs in the presence of an abundant amount of PAA. The established phenomena may be important for understanding the biological effects of polycations.

A polycation causes migration of negatively charged phospholipids from the inner to outer leaflet of the liposomal membrane

Aggregation of the negatively charged liposomes caused by the addition of the linear polycation, poly‐N‐ethyl‐4‐vinylpyridine bromide, was studied. At the point of maximal size and zero electrophoretic mobility of aggregates, the concentration of positive charges brought in by the adsorbed polycation was found to be equal to the total concentration of negative charges both on the outer and inner surface of the lipid bilayer. Since polycation saturation of the liposomal negative charges was found to occur without disruption of the membrane, it was concluded that the polycation induced migration of negatively charged phospholipid molecules from the inner to outer leaflet of the bilayer.

What happens to negatively charged lipid vesicles upon interacting with polycation species

Complexation of synthetic polycations with negative lipid vesicles as cell-mimetic species was studied. It was found that such interaction could be accompanied by lateral lipid segregation, highly accelerated transmembrane migration of lipid molecules (polycation-induced flip-flop), incorporation of adsorbed polycations into vesicular membrane as well as aggregation and disruption of vesicles. A polycation adsorbed on the surface of liquid vesicles due to electrostatic attraction could be completely removed from the membrane by increase in simple salt concentration or by recomplexation with polyanions. In contrast, adsorption of a polycation carrying pendant hydrophobic groups was irreversible apparently due to incorporation of these groups into the hydrophobic part of the vesicular membrane. The above mentioned phenomena were examined depending on the polycation structure, fraction of charged lipids in the membrane, vesicle phase state and ionic strength of solution.

Interaction of a cationic polymer with negatively charged proteoliposomes.

Proteoliposomes were prepared by making bilayer vesicles from neutral egg yolk lecithin and negatively charged alpha-chymotrypsin that had been previously stearoylated. Interaction of these proteoliposomes with a cationic polymer, poly-(N-ethyl-4-vinylpryidinium bromide) (PEVP) was examined. For comparison purposes, interaction of PEVP with egg lecithin vesicles containing an anionic phospholipid, cardiolipin, was also examined. Binding of PEVP to both types of vesicles was electrostatic in nature with the polymer manifesting a higher affinity to the cardiolipin relative to the enzyme. PEVP had no effect on the permeability of the bilayer membranes to sodium chloride. On the other hand, PEVP increased the transmembrane permeability of the nonionic anti-tumor drug, doxorubicin. The greater the negatively charged component in the membrane, the greater the PEVP effect. Polycation binding to the vesicles was accompanied by clustering of the stearoylated chymotrypsin (sCT) molecules within the membrane. This protein clustering is most likely responsible for the increase in the doxorubicin permeation. Enzymatic activity of the membrane-associated sCT remained unchanged upon PEVP binding. These findings seem relevant to the effects of polyelectrolytes on cellular membranes.

The cooperation characteristics between polyelectrolytes during their reactions

A series of polyelectrolyte (PEL) systems have been used as examples to show that the conversion as a function of pH is a standard function over a wide range of initial PEL concentrations c0, and is independent of the latter. Electron microscopy and the rapid sedimentation method have been used to study the compositions of the complexes. The conclusion is reached that the reactions between PEL take place in autonomous micro-reactors.

Catalytic properties and conformation of hydrophobized α‐chymotrypsin incorporated into a bilayer lipid membrane

A set of artificially hydrophobized α‐chymotrypsin derivatives, carrying 2–11 stearoyl residues per enzyme molecule, were synthesized and their catalytic parameters and conformation in water solution and in the liposome‐bound state were investigated. Hydrophobization of α‐chymotrypsin and its further incorporation into phosphatidylcholine (PC) liposomes have no effect on the rate constant of the N‐acetyl‐L‐tyrosine ethyl ester (ATEE) ester bond hydrolysis (k cat). At the same time, an increase in the number of stearoyl residues attached to the enzyme results in a drastic decrease of ATEE binding to the active center (K M increase). Incorporation of the hydrophobized enzyme into the PC liposome membrane results in K M recovery to nearly that of native α‐chymotrypsin. The above changes are accompanied by partial unfolding of the enzyme molecules observed by fluorescence measurements. The obtained results are of interest to mimic the contribution of surface hydrophobic sites in the functioning of membrane proteins.

Substitution reactions in ternary systems of macromolecules

Abstract Substitution reactions were investigated on a macromolecular system containing two different types of poly-anions, namely sodium polymethacrylate (A), sodium polyethylene sulphonate (B), and the poly-cations of poly-4-vinyl-N-ethylpyridinium bromide (C). The reaction was carried out by adding to the water-soluble non-stoichiometric poly-electrolytic complex (PEC) portions of the competing polymer: PEC(AC) +B⇆PEC(BC)+A (1). Reaction (1) can have two mechanisms depending on the reaction conditions; firstly dissociation of PEC(AC) will cause the C-parts to enter the solution where their bonding of the B macromolecules will yield PEC(BC), and secondly by an electrostatic reaction of the B-parts with the ionic groups of C present in the defects (loops) of PEC(AC) to form mixed ternary PEC which are then transformed to PEC(BC) due to phase-separation of the solutions. Variation of the reaction (1) conditions (addition of a low mol.wt. electrolyte, a change of the length of the polymeric reagents, etc.) can result in an effective control of the substitution route in one or the other direction. The general validity of the process types described is shown.