Hybrids of Polymer Multilayers, Lipids, and Nanoparticles: Mimicking the Cellular Microenvironment.

Here we address research directions and trends developed following novel concepts in 2D/3D self-assembled polymer structures established in the department led by Helmuth Möhwald. These functional structures made of hybrids of polymer multilayers, lipids, and nanoparticles stimulated research in the design of the cellular microenvironment. The composition of the extracellular matrix (ECM) and dynamics of biofactor presentation in the ECM can be recapitulated by the hybrids. Proteins serve as models for protein-based biofactors such as growth factors, cytokines, hormones, and so forth. A fundamental understanding of complex intermolecular interactions and approaches developed for the externally IR-light-triggered release offers a powerful tool for controlling the biofactor presentation. Pure protein beads made via a mild templating on vaterite CaCO3 crystals can mimic cellular organelles in terms of the compartmentalization of active proteins. We believe that an integration of the approaches developed and described here offers a strong tool for engineering and mimicking both extra- and intracellular microenvironments.

Structure of the complex of cytochrome c with cardiolipin in non-polar environment.

The complex of mitochondrial protein cytochrome c (CytC) with anionic phospholipid cardiolipin (CL) plays a crucial role in the initiation of apoptosis by catalyzing lipid peroxidation in mitochondrial membranes. In our previous papers, we found that CytC and CL mixed in millimolar concentrations form a sediment showing microcrystals composed of nanospheres (Cyt-CL) of 11-12 and 8 nm in diameter. The hypothesis was proposed that Cyt-CL, having hydrophobic shell, may appear inside the membrane lipid bilayer in mitochondria and peroxidase membrane phospholipids so initiating the apoptotic cascade. In this work, Cyt-CL complex dissolved in chloroform or hexane was investigated as a model of the complex in mitochondrial membranes. We used dynamic light scattering method to measure the size of the particles. The analysis of particles size distribution of Cyt-CL in chloroform allows to reveal three dominant diameters of 12.1 ±â€¯1.4, 7.8 ±â€¯1.0, and 4.7 ±â€¯0.7 nm. The first two values are closed to those, earlier obtained with small-angle X-ray scattering method in Cyt-CL microcrystals, 11.1 ±â€¯1.0 and 8.0 ±â€¯0.7 nm. CL extracted in chloroform-methanol forms a real solution of particles with diameter of 0.7 ±â€¯0.1 nm. In methanol-water phase, CL and CL + CytC mixture form particles of 83.7 ±â€¯9.8 and 71.3 ±â€¯11.6 nm, respectively. Apparently, cardiolipin in 50% methanol forms single-layer liposomes regardless of the presence of CytC in the medium. Partial unfolding of CytC in the complex was evidenced by (a) appearance of fluorescence of tyrosine and tryptophan residues and (b) disappearance of the absorption band at 699 nm due to breakdown of heme iron - methionine bond > F⋯S(Met80). In hydrophobic solvent Cyt-CL exhibited quasi-lipoperoxidase and lipoxygenase activity as was shown in kinetic measurements of chemiluminescence enhanced by coumarin C-525, a selective sensitizer of chemiluminescence, associated with reactions of lipid peroxyl radicals. Our data in this model system do not contradict the hypothesis (Vladimirov, Y.A. et al. Biochemistry (Mosc) 78, 1086-1097) that nanospheres of Cyt-CL complex, embedded into the lipid phase of mitochondrial membrane, catalyze lipid peroxidation, thereby initiating apoptosis.

The mechanism of catalase loading into porous vaterite CaCO3 crystals by co-synthesis.

Porous vaterite CaCO3 crystals are nowadays extensively used as high-capacity bio-friendly sacrificial templates for the fabrication of such protein-containing nano- and micro-particles as capsules and beads. The first step in the protein encapsulation is performed through loading of the protein molecules into the crystals. Co-synthesis is one of the most useful and simple methods proven to effectively load crystals with proteins; however, the loading mechanism is still unknown. To understand the mechanism, in this study, we focus on the loading of a model protein catalase into the crystals by means of adsorption into pre-formed crystals (ADS) and co-synthesis (COS). Analysis of the physico-chemical characteristics of the protein in solution and during the loading and simulation of the protein packing into the crystals are performed. COS provides more effective loading than ADS giving protein contents in the crystals of 20.3 and 3.5 w/w%, respectively. Extremely high loading for COS providing a local protein concentration of about 550 mg mL-1 is explained by intermolecular protein interactions, i.e. formation of protein aggregates induced by CaCl2 during the co-synthesis. This is supported by a lower equilibrium constant obtained for COS (5 × 105 M-1) than for ADS (23 × 105 M-1), indicating a higher affinity of single protein molecules rather than aggregates to the crystal surface. Fitting the adsorption isotherms by classical adsorption models has shown that the Langmuir and BET models describe the adsorption phenomenon better than the Freundlich model, proving the aggregation in solution followed by adsorption of the aggregates into the crystals. We believe that this study will be useful for protein encapsulation through CaCO3 crystals using the COS method.

Cytochrome c-Cardiolipin Complex in a Nonpolar Environment.

Programmed cell death (apoptosis) plays an important role in the life of multicellular organisms and in the development of socially significant human diseases. Cytochrome c-cardiolipin complex (Cyt-CL) is formed at the very beginning of a cascade of apoptotic reactions. Nevertheless, the structure of the complex and the mechanism of its participation in lipid peroxidation in mitochondrial membranes are not yet understood. In previous work (Vladimirov, Y. A., et al. (2011) Crystallography, 56, 712-719), it was shown that the Cyt-CL complex precipitates in concentrated water solution, the sediment containing orderly nanospheres formed by cytochrome c molecules with changed conformation and surrounded by a cardiolipin monolayer, and they are essentially hydrophobic. In this work, we obtained chloroform and hexane solutions of Cyt-CL with lipid/protein ratio of 77 ± 11. The conditions are described under which the solutions were obtained. Study of the properties of Cyt-CL solutions in hydrophobic media will reveal their structure and the mechanism of their catalytic activity inside the lipid layer of biological membranes.

Temperature-induced molecular transport through polymer multilayers coated with PNIPAM microgels.

Polyelectrolyte multilayers serve as effective reservoirs for bioactive molecules which are stored and released from the multilayers for cellular applications. However, control over the release without significantly affecting the multilayers and biomolecules is still a challenge. On the other hand, externally stimulated release would make the multilayers promising for the development of stimuli-sensitive planar carriers with release performance switched on demand. In this study soft composite films are designed by coating hyaluronic acid/poly-l-lysine (HA/PLL) multilayers with temperature responsive poly(N-isopropylacrylamide) (PNIPAM) microgels. Microgels are flattened and immersed into the multilayers to maximize the number of contacts with the surrounding polyelectrolytes (HA and PLL). The microgel coating serves as an efficient switchable barrier for the PLL transport into the multilayers. PLL diffusion into the film is significantly hindered at room temperature but is dramatically enhanced at 40 °C above the volume phase transition temperature (VPTT) of PNIPAM at 32 °C associated with microgel shrinkage. Scanning force microscopy micrographs show that the mechanism of volume phase transition on soft surfaces cannot be directly deduced from the processes taking place at solid substrates.

LC/MS analysis of cardiolipins in substantia nigra and plasma of rotenone-treated rats: Implication for mitochondrial dysfunction in Parkinson's disease.

Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathologic features of Parkinson's disease (PD). As rotenone acts as an inhibitor of mitochondrial respiratory complex I, we employed oxidative lipidomics to assess oxidative metabolism of a mitochondria-specific phospholipid, cardiolipin (CL), in substantia nigra (SN) of exposed animals. We found a significant reduction in oxidizable polyunsaturated fatty acid (PUFA)-containing CL molecular species. We further revealed increased contents of mono-oxygenated CL species at late stages of the exposure. Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily "oxidizable" fatty acid residues (arachidonic and docosahexaenoic acids) remained non-oxidized. Elevated levels of PUFA CLs were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified CL molecular species in SN and detection of PUFA-containing CL species in plasma may contribute to better understanding of the PD pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic 'eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2.

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.