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.

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.

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.