Nickolay S. Melik-Nubarov

Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin

The effects of ethylene oxide and propylene oxide block copolymers (pluronics) on the permeability of several weak acids and bases through bilayer lipid membranes have been studied by the methods of monitoring (1) pH shifts near planar bilayers, (2) doxorubicin fluorescence quenching inside liposomes, and (3) current transients in the presence of hydrophobic anions. It has been shown that pluronics facilitate the permeation of comparatively large molecules (such as 2-n-undecylmalonic acid and doxorubicin) across lipid bilayers, while the permeation of small solutes (such as ammonium and acetic acid) remains unaffected. Pluronics also accelerate the translocation of large hydrophobic anions (tetraphenylborate). The effect of pluronics correlates with the content of propylene oxide units: it is enhanced when the portion of polypropylene oxide block in the copolymer is increased. The action of the pluronic on lipid membrane permeability differs from the effect of the conventional detergent Triton X-100, which does not affect doxorubicin transport if added at concentrations similar to those used for pluronics. It has been proposed that pluronics accelerate the processes of solute diffusion within lipid bilayers (in a structure-dependent manner) rather than influencing the rate of solute adsorption/desorption on the membrane surface. We suppose that the effect of pluronics on doxorubicin permeation across lipid bilayers along with the known effect on the multidrug resistance protein determines its influence on the therapeutic activity of anthracycline drugs.

Porphyrin photosensitizers solubilized with pluronics in the oxidation of tryptophan

It was shown that water-insoluble porphyrins solubilized with pluronics (ternary block copolymers of ethylene oxide and propylene oxide) efficiently transfer photoexcitation energy to molecular oxygen dissolved in aqueous media to excite it into the singlet state, which is active in oxidizing organic substrates. It was established that the degree of solubilization of porphyrin photosensitizers (PPSs) γ defined as the proportion of PPS molecules passed into the aqueous phase in the dissolution of formed films depends on a single parameter, the initial ratio between the porphyrin and pluronic molar concentrations q. γ = γ(q) dependences for the solubilization of dissimilar water-insoluble porphyrins were analyzed. It was shown that the behavior of all the obtained γ = γ(q) dependences is changed at a characteristic value qmax: when q š qmax, γ ∼ 1, and, when q ≥ ?qmax, the degree of solubilization decreases. It was concluded that solubilized porphyrins are efficient photosensitizers in the oxidation of organic substrates (tryptophan).

Complexes of Chlorin e6 with Pluronics and Polyvinylpyrrolidone: Structure and Photodynamic Activity in Cell Culture

Polymeric carriers are extensively used in photodynamic therapy (PDT) for increase of efficacy of photosensitizers. Here, we report the influence of nine Pluronic copolymers on phototoxicity of chlorin e6 (Ce6), in particular 5‐ to 7‐fold rise in the phototoxicity caused by hydrophilic Pluronics F127, F108, F68 and F87 and practically no influence on Ce6 of more hydrophobic polymers. The revealed value of 0.2 mg mL−1 of Pluronic F127 concentration sufficient for half‐of‐maximal increase of Ce6 photodynamic activity proved to be close to 0.16 mg mL−1 inherent in well‐documented carrier poly(N‐vinylpyrrolidone) (PVP). The dissociation constants of Ce6 complexes with Pluronic F127 and PVP that were estimated from UV spectra were 0.252 and 0.036 mg mL−1, respectively, indicating higher stability of Ce6 complex with PVP. According to the results of 1H‐NMR studies of Ce6 complexes, the porphyrin interacts not only with hydrophobic regions but also with hydrophilic sides of both polymers.

Development of novel formulations for photodynamic therapy on the basis of amphiphilic polymers and porphyrin photosensitizers. Porphyrin-polymer complexes in model photosensitized processes

We have studied the effect of amphiphilic polymers with different structure (polyvinylpyrrolidone, polyethyleneoxide and a triblock copolymer of ethylene- and propyleneoxide—(Pluronic F127)) on the photoactivity of a hematoporphyrin derivative (dimegin). It has been shown that such polymers can cause a considerable increase in the porphyrin photosensitizer (PPS) activity both in the process of singlet oxygen photogeneration and in the reaction of a substrate photooxidation in D2O and water. Among the studied polymers, polyvinylpyrrolidone appeared to have a most significant influence onto the photoactivity of dimegin. We attribute the observed effect of the amphiphilic polymers on the photoactivity of dimegin to the presence of polymer-porphyrin interactions resulting in the porphyrin disaggregation in aqueous phase. Using 1H NMR spectroscopy, we have found that dimegin binds to the polymers via the PPS interaction mainly with the hydrophobic fragments of polymeric macromolecules. However, in the case of polyvinylpyrrolidone we observed also PPS interactions with the hydrophilic fragments of macromolecules.

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.

The Control of Membrane Properties by Synthetic Polymers

Abstract This review describes the effects caused by synthetic polymers on model lipid membranes. Among a large variety of synthetic polymers, we focused on two quite different groups. One of them consists of polyelectrolytes, whose adsorption on lipid membranes is driven by Coulomb forces, and the other group consisting of Pluronics and polyethoxylated surfactants that have amphiphilic structure and interact with lipid bilayers by hydrophobic forces. This choice was determined by the continuously increasing interest to the application of these polymers in biomedical research. It was shown that interactions of cationic polymers with oppositely charged lipid bilayers result in the formation of lipid domains in the membranes, influence the lateral diffusion of lipid molecules and proteins incorporated into the bilayer, modulate functional activity of membrane proteins and in some cases even favor asymmetrical distribution of lipids. Incorptions of amphiphilic copolymers into lipid bilayers may result in changes in the rate of flip-flop and membranes microviscosity. Polymer-membrane interactions in both cases cause changes in the membrane premeability, obviously resulting from formation of disturbances in the membrane packing. In case of Pluronics, non-covalent interactions between the membrane-bound polymer and the permeant may also contribute to the transportation of the latter through lipid bilayer. Special attention in this review is paid to the relatioship between the polymer structure and its ability to modulate membrane properties. A qualitative thermodynamic treatment of the polymer-membrane interactions allowed to explain the polymer effects on the bilayer structure and led to a list of properties that the polymer structure must possess in order to affect membrane permeability.

Peroxyoxalate Chemiluminescent Reaction as a Tool for Elimination of Tumour Cells Under Oxidative Stress

The overproduction of hydrogen peroxide is an inherent feature of some tumour cells and inflamed tissues. We took advantage of this peculiarity to eliminate cells using chemiluminescent peroxyoxalate reaction. We designed dispersions containing polyoxalate and tetramethylhematoporhyrin (TMHP) in dimethylphthalate droplets stabilized with Pluronic L64. The porphyrin plays the dual role. On the one hand, it serves as an activator of the peroxyoxalate reaction of polyoxalate with intracellular hydrogen peroxide and experiences excitation as a result of the reaction. The light emitted in the reaction in the model system without cells was used to optimize the dispersion’s composition. On the other hand, TMHP acts as a photosensitizer (PS) causing cell damage. The formation of singlet oxygen led to cell elimination if the dispersions were used in combination with inducers of oxidative stress: hydrogen peroxide, paraquat, antitumour drug doxorubicin, or a nutritional additive menadione. The PS-induced cytotoxicity correlated with the level of intracellular ROS. The developed approach targeted to endogenous ROS is orthogonal to the classical chemotherapy and can be applied to increase its efficiency.

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.