Modeling interaction between gp120 HIV protein and CCR5 receptor.

The study of the process of HIV entry into the host cell and the creation of biomimetic nanosystems that are able to selectively bind viral particles and proteins is a high priority research area for the development of novel diagnostic tools and treatment of HIV infection. Recently, we described multilayer nanoparticles (nanotraps) with heparin surface and cationic peptides comprising the N-terminal tail (Nt) and the second extracellular loop (ECL2) of CCR5 receptor, which could bind with high affinity some inflammatory chemokines, in particular, Rantes. Because of the similarity of the binding determinants in CCR5 structure, both for chemokines and gp120 HIV protein, here we expand this approach to the study of the interactions of these biomimetic nanosystems and their components with the peptide representing the V3 loop of the activated form of gp120. According to surface plasmon resonance results, a conformational rearrangement is involved in the process of V3 and CCR5 fragments binding. As in the case of Rantes, ECL2 peptide showed much higher affinity to V3 peptide than Nt (KD  = 3.72 × 10-8 and 1.10 × 10-6  M, respectively). Heparin-covered nanoparticles bearing CCR5 peptides effectively bound V3 as well. The presence of both heparin and the peptides in the structure of the nanotraps was shown to be crucial for the interaction with the V3 loop. Thus, short cationic peptides ECL2 and Nt proved to be excellent candidates for the design of CCR5 receptor mimetics.

Preparation and characterization of macroporous monoliths imprinted with erythromycin.

The synthesis of macroporous molecularly imprinted monoliths was performed using the monomers system 2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate and erythromycin as a template. The copolymerization was carried out in situ inside 50 mm × 4.6 mm i.d. stainless-steel tubing. The morphology of the monoliths was examined with scanning electron microscopy. The porous characteristics were determined both from the data of hydrodynamic permeability of monoliths and by means of mercury intrusion porosimetry. The retention parameters of target substance (erythromycin), values of calculated imprinting factors and apparent dynamic dissociation constants were obtained for monoliths prepared with the application of different amount of template (4, 8 and 12 mol%). The separations of the mixtures azithromycin/erythromycin and ciprofloxacin/erythromycin were demonstrated. Additionally, the possibility of erythromycin quantification in human blood plasma was shown.

[A multienzyme bioreactor based on a chitinase complex].

A heterogeneous biocatalyst containing a complex of chitinolytic enzymes isolated from the culture medium of bacteria Clostridium paraputrificum on the surface of macroporous monolithic minidisc was obtained. The complex of chitinolytic enzymes was immobilized on the polymer matrix using a multistep method involving the introduction of an intermediate macromolecular spacer. The endochitinase and N-acetylglucosaminidase activity of the heterogeneous biocatalyst was studied.

[Degradation of polyribonucleotides: biocatalysis and the monitoring of products].

Macroporous monolithic material containing covalently linked ribonuclease A was used to create high-performance flow heterogeneous biocatalysts (bioreactors). The kinetic parameters of the degradation of polycytidylic acid were identified, and the properties of the obtained systems were compared. A HPLC method has been developed for monitoring products of biocatalytic degradation of RNA, and the possibility of using biocatalytic and HPLC columns in RNA degradation processes in a multicomponent mixture of biological molecules was shown.

New supramolecular Au(I)-Cu(I) complex as potential luminescent label for proteins.

A novel supramolecular [Au6Cu2(C2C6H4-4-COONC4H4O2)6(Ph2PC6H4PPh2)3](PF6)2 complex functionalized with a succinimide ester alkynyl substituent has been synthesized and characterized using X-ray crystallography, mass spectrometry, and NMR spectroscopy. Like the other complexes of this class, it demonstrates bright emission in acetone and dichloromethane solutions with the excited state lifetime in a microsecond domain. This complex readily reacts with a surface amine group of proteins/enzymes (human serum albumin (HSA), rabbit anti-HSA antibodies, soybean trypsin inhibitor, and α-chymotrypsin) to give covalent conjugates, which contain up to five molecules of the luminescent label bound to the biomolecule. The conjugates keep a high level of the phosphorescent label emission, but in contrast to the parent complex molecule, display excellent solubility and high stability in physiological media. Investigation of the biological activity of the conjugates also showed that the specific structure of the biomolecules remained nearly unchanged upon bonding with the label, which is indicative of a very prospective of the conjugates application in biomolecular detection.

Biocatalytic reactors based on ribonuclease A immobilized on macroporous monolithic supports.

Immobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined.

Polymer monoliths as efficient solid phases for enzymatic polynucleotide degradation followed by fast HPLC analysis.

Two ribonuclease A bioreactors based on lab-made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free-radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to the solid surface via direct covalent binding, while in the second bioreactor the flexible-chain synthetic polymer was used as an intermediate spacer. The effect of temperature, substrate flow rate, and loaded sample volume on the biocatalytic efficiency of the immobilized enzyme was examined. The kinetic parameters of the enzymatic degradation of synthetic polycytidylic acid were calculated and compared to those found for hydrolysis with soluble ribonuclease A. The monitoring of substrate splitting was carried out by means of fast anion-exchange HPLC on an ultra-short monolithic column (disk) using off- and on-line analytical approaches.

[In vitro modeling of cell-scaffold interaction].

The simple approach for modeling of surface ligand - cell receptor interaction is proposed to control the effectiveness of peptide acceptor selected to be immobilized on a scaffold surface in order to promote specific cell adhesion and their subsequent proliferation and bone tissue formation. For experimental realization of such approach the affinity chromatography with use of macroporous monolithic sorbent is suggested. The biospecific GRGDSP-peptide performed the role of scaffold surface ligand which is responsible for cell adhesion, while the "cells" were simulated by polymer (polystyrene) micro particles with EDYPVDIYYLM-DLSYSMKDD-peptide immobilized on their surface. The latter peptide is the integrin molecule active site which is responsible for RGD-sequence binding. Thus the ultra-short monolithic chromatography columns (CIM-disks) represent the simplified model of a scaffold possessing biospecific properties. The qualitative evaluation of complement interaction parameters was performed via frontal analysis method followed by adsorption isotherm plotting and subsequent linearization and mathematical treatment. The data obtained reliably indicate the highly specific character of biological pair binding. This was in a good accordance with results obtained in the cell culture experiments.

Macroporous Polymer Monoliths for Affinity Chromatography and Solid-Phase Enzyme Processing.

Nowadays, monolithic stationary phases, because of their special morphology and enormous permeability, are widely used for the development and realization of fast dynamic and static processes based on the mass transition between liquid and solid phases. These are liquid chromatography, solid-phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including the bioaffinity mode, represents unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In the edited chapter, the examples of the application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by the combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents the original and practically valuable method that can be used in biotechnology. In addition, macroporous monoliths were adapted to the immobilization of such different enzymes as polynucleotide phosphorylase, ribonuclease A, α-chymotrypsin, chitinolytic biocatalysts, ß-xylosidase, and ß-xylanase. The possibility of use of immobilized enzyme reactors based on monoliths for different purposes is demonstrated.

Study of dynamic adsorption behavior of large-size protein-bearing particles.

The subject of this paper is an investigation of the peculiarities of dynamic adsorption behavior of nanoparticles. For this purpose, virus-mimicking synthetic particles bearing different proteins at their outer surface were specially constructed using two approaches, e.g. the cross-linking of proteins and modification of polystyrene microsphere surface by proteins. Two chromatographic modes, namely ion-exchange and affinity liquid chromatography on ultra-short monolithic columns [Convective Interaction Media (CIM) DEAE and CIM QA disks] have been used as a tool for dynamic adsorption experiments. Such parameters as maximum adsorption capacity and its dependence on applied flow rate were established and compared with those obtained for individual proteins. Similarly to individual proteins, it was shown that the maximum of adsorption capacity was not changed at different flow rates. In addition, the permeability of porous space of used monolithic sorbents appeared to be sufficient for efficient separation of large particles and quite similar to the well-studied process applied for individual proteins.

Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex.

The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

Macroporous monoliths for biodegradation study of polymer particles considered as drug delivery systems.

Nanostructures based on biodegradable polymers are often considered as drug delivery systems. The properties of these nanomaterails towards in vitro biodegradation are very important and usually are studied using the model physiological conditions. In this work the novel approach based on application of monolithic immobilized enzyme reactors (IMERs) as the systems for biodegradation study of the nanoobjects of different nature and morphology was suggested. Rigid nanospheres based on poly(lactic acid) and self-assembled nanoobjects formed from block-copolymer of glutamic acid and phenylalanine were applied as model nanomaterials. For that, two enzymes, namely, esterase and papain were chosen for preparation of the monolithic IMERs. The properties of immobilized enzymes were compared to those obtained for soluble biocatalysts in the reaction of poly(lactic acid) and poly(glutamic acid) degradation. The monitoring of substrate destruction process was carried out using different HPLC modes (anion-exchange, cation-exchange or precipitation-redissolution based process) also based on application of the same modern stationary phase, namely, macroporous monoliths (CIM disks and lab-made column). Finally, the applicability of monolithic immobilized enzyme reactors for degradation of polyester and polypetide-based particles was demonstrated and compared to the process observed in human blood plasma.

Self-assembled polypeptide nanoparticles for intracellular irinotecan delivery.

In this research poly(l-lysine)-b-poly(l-leucine) (PLys-b-PLeu) polymersomes were developed. It was shown that the size of nanoparticles depended on pH of self-assembly process and varied from 180 to 650nm. The biodegradation of PLys-b-PLeu nanoparticles was evaluated using in vitro polypeptide hydrolysis in two model enzymatic systems, as well as in human blood plasma. The experiments on the visualization of cellular uptake of rhodamine 6g-loaded and fluorescein-labeled nanoparticles were carried out and the possibility of their penetration into the cells was approved. The cytotoxicity of polymersomes obtained was tested using three cell lines, namely, HEK, NIH-3T3 and A549. It was shown that tested nanoparticles did not demonstrate any cytotoxicity in the concentrations up to 2mg/mL. The encapsulation of specific to colorectal cancer anti-tumor drug irinotecan into developed nanocontainers was performed by means of pH gradient method. The dispersion of drug-loaded polymersomes in PBS was stable at 4°C for a long time (at least 1month) without considerable drug leakage. The kinetics of drug release was thoroughly studied using two model enzymatic systems, human blood serum and PBS solution. The approximation of irinotecan release profiles with different mathematical drug release models was carried out and allowed identification of the release mechanism, as well as the morphological peculiarities of developed particles. The dependence of encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The maximal drug loading was found as 320±55µg/mg of polymersomes. In vitro anti-tumoral activity of irinotecan-loaded polymersomes on a colon cancer cell line (Caco-2) was measured and compared to that for free drug.

Molecularly imprinted macroporous monoliths for solid-phase extraction: Effect of pore size and column length on recognition properties.

The series of macroporous monolithic molecularly imprinted monoliths differed by pore size, column length (volume) and amount of template used for imprinting was synthesized using methacrylic acid and glycerol dimethacrylate as co-monomers and antibiotic ciprofloxacin as a template. The prepared monoliths were characterized regarding to their permeability, pore size, porosity, and resistance to the flow of a mobile phase. The surface morphology was also analyzed. The slight dependence of imprinting factor on flow rate, as well as its independence on pore size of macroporous molecularly imprinted monolithic media was observed. The column obtained at different conditions exhibited different affinity of ciprofloxacin to the imprinted sites that was characterized with Kdiss values in the range of 10(-5)-10(-4)M. The solid-phase extraction of ciprofloxacin from such biological liquids as human blood serum, human urine and cow milk serum was performed using the developed monolithic columns. In all cases, the extraction was found to be 95.0-98.6%. Additionally, the comparison of extraction of three fluoroqinolone analogues, e.g. ciprofloxacin, levofloxacin and moxifloxacin, from human blood plasma was carried out. Contrary to ciprofloxacin extracted with more than 95%, this parameter did not exceed 40% for its analogues.

Detection of human genome mutations associated with pregnancy complications using 3-D microarray based on macroporous polymer monoliths.

Analysis of variations in DNA structure using a low-density microarray technology for routine diagnostic in evidence-based medicine is still relevant. In this work the applicability of 3-D macroporous monolithic methacrylate-based platforms for detection of different pathogenic genomic substitutions was studied. The detection of nucleotide replacements in F5 (Leiden G/A, rs6025), MTHFR (C/T, rs1801133) and ITGB3 (T/C, rs5918), involved in coagulation, and COMT (C/G, rs4818), TPH2 (T/A, rs11178997), PON1 (T/A rs854560), AGTR2 (C/A, rs11091046) and SERPINE1 (5G/4G, rs1799889), associated with pregnancy complications, was performed. The effect of such parameters as amount and type of oligonucleotide probe, amount of PCR product on signal-to-noise ratio, as well as mismatch discrimination was analyzed. Sensitivity and specificity of mutation detections were coincided and equal to 98.6%. The analysis of SERPINE1 and MTHFR genotypes by both NGS and developed microarray was performed and compared.

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA).

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.

In vitro comparison of complementary interactions between synthetic linear/branched oligo/poly-L-lysines and tissue plasminogen activator by means of high-performance monolithic-disk affinity chromatography.

The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-L-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.

Affinity chromatography of proteins on monolithic columns.

At present, monolithic stationary phases, because of their morphology, are widely used for development and realization of fast dynamic and static processes based on mass transition between liquid and solid phases. These are liquid chromatography, solid phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including bioaffinity mode, represents a unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In this work, the examples of application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator from E. coli cell lysate and Chinese Hamster Ovary cell culture supernatant, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents an original and practically valuable method that can be used in biotechnology.

Methacrylate-based monolithic layers for planar chromatography of polymers.

A series of macroporous monolithic methacrylate-based materials was synthesized by in situ free radical UV-initiated copolymerization of functional monomers, such as glycidyl methacrylate (GMA), butyl methacrylate (BuMA), 2-aminoethyl methacrylate (AEMA), 2-hydroxyethyl methacrylate (HEMA) and 2-cyanoethyl methacrylate (CEMA), with crosslinking agent, namely, ethylene glycol dimethacrylate (EDMA). The materials obtained were applied as the stationary phases in simple and robust technique - planar chromatography (PLC). The method of separation layer fabrication representing macroporous polymer monolith bound to the specially prepared glass surface was developed and optimized. The GMA-EDMA and BuMA-EDMA matrixes were successfully applied for the separation of low molecular weight compounds (the mixture of several dies), as well as poly(vinylpyrrolidone) and polystyrene homopolymers of different molecular weights using reversed-phase mechanism. The materials based on copolymers AEMA-HEMA-EDMA and CEMA-HEMA-EDMA were used for normal-phase PLC separation of 2,4-dinitrophenyl amino acids and polystyrene standards.

Monolithic bioreactors: effect of chymotrypsin immobilization on its biocatalytic properties.

The effect of different modes of alpha-chymotrypsin attachment to the surface of methacrylate-based ultrashort monolithic minicolumns on enzyme activity has been studied. The immobilization of protease was carried out via direct covalent binding of chymotrypsin, as well as via its attachment through small and polymer spacers. It was established that the lowest enzyme activity against N-benzoyl-l-tyrosine ethyl ester was found for bioreactor obtained via direct attachment of chymotrypsin to the surface of GMA-EDMA minidisks, whereas the highest parameter close to that determined for dissolved enzyme was found in the case of bioreactor prepared by the introduction of copolymer of 2-deoxy-N-methacryloylamido-d-glucose with N-vinylpyrrolidone and acrolein as a long and flexible polymer spacer. Additionally, the effect of flow rate of substrate recirculation on bioconversion efficiency was examined. Independently on immobilization method, the increase of flow rate led to the raise of biocatalytic efficiency.