Advances and Main Results

NANOMATERIALS IN BIOLOGY AND MEDICINE

The collaborative network between:

- Department of General Biophysics, University of Lodz, Lodz, Poland,

- Departamento de Química Inorgánica, Universidad de Alcalá, Alcalá de Henares, Spain,

- Laboratoire de Chimie de Coordination, CNRS, Toulouse, France, and

- Institute of Chemical Biology and Experimental Medicine of SB of RAS, Novosibirsk, Russia,

- Laboratory of Proteomics, Institute of Biophysicvs and Cell Engineering of NASB, Minsk, Belarus.

.

  • Dendrimers and fluorescent probes

We have provided the model studies on binding different substances by carbosilane, phosphorus, poly-lysine, polyamidoamine (PAMAM) and poly(propylene imine) (PPI) dendrimers. The ability of above mentioned dendrimers to bind substances was estimated by their interaction with fluorescent probes (8-Anilino-1-naphthalenesulfonate (ANS), dansylamide, dansylsarcosine). Based on Scatchard-Klotz equations the binding capacity of dendrimers was calculated and the technique for estimation of the constants of binding and the number of binding centers of dendrimers was proposed. We found that dendrimers compete with serum albumins in fluorescent probe ANS binding and proposed that dendrimers can bind endogenous and exogenous toxins in a body.

  • Dendrimers and toxins (heavy metals, bilirubin, hippuric acid)

To confirm this hypothesis we studied interactions between PAMAM dendrimers and some endogenous and exogenous toxins in vitro. Exogenous toxins were ions of heavy metals: Cd2+ and Cs2+, endogenous toxins were bilirubin, hippuric acid, deoxycholic acid. It was found that PAMAM dendrimers are able to bind ions of heavy metals. This kind of interaction significantly decreases the impact of ions on serum proteins. Also we showed that PAMAM dendrimers to bind bilirubin and hippuric acid that indicates the possibility of dendrimers’ application for decrease of bilirubin level at hyperbilirubinemia and hepatitides.

  • Dendrimers and proteins

The complex analysis of interaction between dendrimers and proteins was elucidated. It was found that the impact of PAMAM dendrimers on human and serum albumins depends on pH and ionic strength of solution and was maximal at low values of ionic strength and pH 7.4. Studying interaction between cationic PAMAM dendrimers and human and bovine serum albumins showed that albumins have at least 5 non-specific binding sites for the molecules of cationic dendrimers. Also, we investigated the interaction between PAMAM dendrimers and bovine serum albumin (free of fatty acids, loaded with oleic acid, linoleic acid, oleic+linoleic acids, oleic+linoleic+arachidonic acids). It was found that the presence of loaded fatty acids decreases dendrimer-protein interaction. The interaction between albumin loaded with several fatty acids and dendrimer led to release of fatty acids from albumin macromolecule. The interaction between PAMAM dendrimers and γ-globulin, alkaline phosphatase, azurin, aldolase, RNaseT1, alcohol dehydrogenase, human serum albumin was studied by room temperature phosphorescence and fluorescence techniques. The differences in the impact of dendrimers on protein structure were found to be depended on dendrimer end groups (cationic, neutral, anionic), protein charge, internal viscosity, intramolecular dynamics. The decrease of protein enzymatic activity upon addition of dendrimers was also observed.

  • Dendrimers as gene carriers for anti-HIV antisense oligodeoxynucleotides and siRNA against human immunodeficiency virus

The analysis of carbosilane and phosphorus dendrimers as gene carries for anti-HIV antisence oligodeoxynucleotides (ODN) and siRNA delivery was performed. The charge ratio of dendrimers and ODNs/siRNA to form neutrally charged dendriplexes was determined. The sizes of dendriplexes vary between 100 and 400 nm. Such complexes are stable within 48 hours while using stable dendrimers and are stable in 1-3 hours while self-destroying dendrimers use. Dendriplexes with siRNA remained undestroyed after RNAse1 exposure. Attachment of short nucleotides to the dendrimers led to significant decrease of their interaction with serum albumins.

The results on transfection showed that introduction of dendriplexes with both ODNs and siRNA significantly decreases HIV1 virus replication in peripheral blood mononuclear cells and the lymphocytic cell line SupT1.

  • Dendrimers for delivery of anti-cancer drugs

The analysis of PAMAM and phosphorus dendrimers as drug carries for delivery commercial anti-cancer drug cisplatin showed that dendrimers significantly (10-fold) increase the anti-tumor effect of cisplatin. In combination with dendrimers the applied concentration of cisplatin to achieve appropriate toxic effect in tumor cells can be reduced in 10 times.

PROTEOMICS

  • The new biophysical mechanism of regulating the proteome activity under normal condition and under pathology realized by transformating a structural-dynamic state of proteins against the background of their structure constancy was established.
  • The scheme of interralation between internal dynamics, conformation and functional activity of protein was proposed. It was established that gradual changes in the protein activity are achieved by the shifts of internal dynamics and the possibility of gradual regulation of protein function is provided by the conformational transitions. The existence of optimal amplitude and frequency of equilibrium motions of protein structural elements for protein functioning was shown.
  • The fluctuation model of enzyme-substrate interactions was proposed. In contrast to generally accepted models of “key-lock” by Fisher and “induced correspondence” by Koshland, the fluctuation model is based on taking into consideration the role of the protein internal dynamics in the processes of substrate binding by the active site of enzyme, destabilization of covalent bonds of substrate and release of reaction products.
  • New mechanisms of ion strength and pH action on enzyme activity realized by the shifts in protein slow internal dynamics were detected.
  • The conception about the ability of proteins to exist in several thermodynamically stable and partially folded states differing in amplitudes of structural transformations and the modification degree of functional activity was stated and experimentally substantiated.
  • New method to study the slow (millisecond) internal dynamics of membrane protein structure in situ was developed. The method is based on recording of parameters of tryptophane phosphorescence at room temperature of proteins. The apparatus to measure the phosphorescence (automatic tau-phosphorimeter and spectraphosphorimeter) was developed.
  • Contrasting differences in slow internal dynamics of peripheral and integral membrane proteins of erythrocyte membranes in situ were detected. The effect of protein-protein and protein-lipid interactions on slow internal dynamics of membrane protein structure was revealed.
  • It was shown that limited slow internal dynamics of membrane proteins as compared with the majority of water-soluble proteins is caused by the presence of protein ensembles in the membrane, isolation of macromolecules from aqueous surroundings in lipid bilayer composition and by high content of α-helices and β-sheets in macromolecules.
  • The possibility of physical and chemical factors and agents to modify the slow internal dynamics of membrane proteins in human erythrocytes was revealed.
  • It was shown that changes in the slow internal dynamics of proteins along with their conformational rearrangements play an important role in the processes of intracellular signaling and transformation of external signals to the cell functional response.
  • The relation was revealed between the changes in slow internal dynamics of membrane proteins and development of pathology. The changes in the structural and dynamic state of lymphocyte membrane proteins of patients with oncological and autoimmune diseases were revealed. It was shown that aggregation of proteins of cataract crystalline lens tissues is associated with rise in inflexibility of macromolecule structure.
  • The new method for analysis lipid peroxidation products in the biological membranes in situ was developed. The method is based on recording spectral and kinetic phosphorescence parameters of lipid peroxidation products at room temperature. Unlike the existing methods the phosphorescence method makes it possible to carry out monitoring of lipid peroxidation product accumulation at different sites of biological membranes.
  • It was established that accumulation of lipid peroxidation products in biological membranes begins locally in the inter-proteins areas of lipid matrix. Then the accumulation area of lipid peroxidation products extends to annular lipids. At the next stage, the products of lipid peroxidation are immobilized in near-protein areas and on proteins with formation of Schiff’s bases.
  • It was shown that the development of pathology associated with the accumulation of lipid peroxidation products in membranes begins at the stage of immobilization of lipid peroxidation products in the areas of annular lipids and proteins.
  • Inhibitory analysis of thrombin, trypsin and α-chymotrypsin action at their low concentrations on aggregation activity of platelets under conditions of blocking phospholipase C, cyclooxygenase, lipoxygenase and calmodulin-dependent enzymes was carried out. It was shown that products of phosphoinositides hydrolysis and arachidonic acid metabolism are involved in processes of platelets aggregation under the action of proteolytic enzymes.

Top