Correlative Fluorescent Scanning Probe Nanotomography Used to Study the Intracellular Distribution of Doxorubicin in MCF-7 Human Breast Adenocarcinoma Cells.

Developing technologies for efficient targeted drug delivery for oncotherapy requires new methods to analyze the features of micro- and nanoscale distributions of antitumor drugs in cells and tissues. A new approach to three-dimensional analysis of the intracellular distribution of cytostatics was developed using fluorescence scanning optical-probe nanotomography. A correlative analysis of the nanostructure and distribution of injected doxorubicin in MCF-7 human breast adenocarcinoma cells revealed the features of drug penetration and accumulation in the cell. The technology is based on the principles of scanning optical probe nanotomography and is applicable to studying the distribution patterns of various fluorescent or fluorescence-labelled substances in cells and tissues.

Effect of lipopeptide structure on gene delivery system properties: Evaluation in 2D and 3D in vitro models.

Development of efficient biodegradable, environmentally responsive, biocompatible and non-toxic delivery system is needed for efficient gene delivery. As well known, properties of the vehicle are determined by the structure of carrier components. The aim of the current study was to estimate in vitro transfection efficacy of aliphatic di-, tri- and tetrapeptide-based cationic lipoplexes loaded with siRNA in function of a number of cationic groups using 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Physicochemical properties and cytotoxicity of the liposomes were found to be dependent upon a number of amino acid derivatives in an amphiphilic polar head. Uptake of liposomes loaded with nucleic acid (lipoplexes) and their localization in HEK293T cells was studied by confocal microscopy. The liposomes based on lipotripeptides had the highest transfection efficiency which was 20-fold higher than those fabricated from lipotetrapeptides.

[Biodegradablescaffolds based on chitosan: Preparation, properties, and use for the cultivation of animal cells].

The influence of the conditions of the formation of chitosan hydrogels crosslinked with glutaraldehyde (GA) or genipin (the polysaccharide molecular weight, pH level, and concentration of the chitosan solution) on the gel time and the properties of biopolymer scaffolds for tissue engineering obtained by the freeze-drying of hydrogels was studied. The resulting scaffolds had different structures (morphology, degree of anisotropy, average pore size) and moisture-retaining capacities. The cytotoxicity of biodegradable scaffolds based on chitosan with a low content of genipin and GA was studied for the first time. Using the L929 mouse fibroblasts model line, we demonstrated that scaffolds based on chitosan with a molecular weight of 320 and 190 kDa crosslinked with genipin and GA (0.005 and 0.01 mol/mol of chitosan amino groups) are biocompatible. Using confocal laser microscopy, we demonstrated that the cells are uniformly distributed in all scaffold samples and they successfully grew and proliferated when cultured in vitro for 4 days.

Polylactide-based microspheres prepared using solid-state copolymerized chitosan and d,l-lactide.

Amphiphilic chitosan-g-poly(d,l-lactide) copolymers have been manufactured via solid-state mechanochemical copolymerization and tailored to design polyester-based microspheres for tissue engineering. A single-step solid-state reactive blending (SSRB) using low-temperature co-extrusion has been used to prepare these copolymers. These materials have been valorized to stabilize microspheres processed by an oil/water emulsion evaporation technique. Introduction of the copolymers either in water or in the oil phase of the emulsion allowed to replace a non-degradable emulsifier typically used for microparticle preparation. To enhance cell adhesion, these copolymers were also tailored to bring amino-saccharide positively charged segments to the microbead surface. Size distribution, surface morphology, and total microparticle yield have been studied and optimized as a function of the copolymer composition.

[Delta-sleep inducing peptide entrapment and release from polymer hydrogels based on modified polyvinyl alcohol in vitro].

The aim of the study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to evaluate peptide release kinetics from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels on the basis of poly(vinyl alcohol) acrylic derivative (Acr-PVA) as well as macroporous hydogels containing epoxy groups which were synthesized by copolymerization of this monomer with glycidyl methacrylate. The isotropic hydrogels were fabricated at positive temperatures while the macroporous hydrogels (cryogels) were prepared at the temperatures below zero. The peptide was entrapped into macroporous modified PVA hydrogels by addition of a peptide solution on previously fabricated matrices, while into PVA-GMA hydrogels containing epoxy groups peptide immobilization was carried out by incubation of hydrogel matrices in the peptide solution. In the case of isotropic hydrogels the peptide was added into the polymer mixture at a hydrogel formation reaction. The peptide release kinetics was studied by incubation of hydrogels in PBS (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by phase-reverse HPLC. DSIP release from the macroporous PVA hydrogel after 30 min incubation was 74, 70 and 64% in water, PBS and 0.9% NaCl, relatively, and it was completed in 3 hs. From the isotropic hydrogel the release neither peptide nor products of its degradation was not observed even after 48 hs of incubation. For freshly prepared hydrogel the release kinetics was as follows: 27 and 78% in 30 and 33 hs, relatively. In the case of the lyophilized hydrogel samples the peptide release was 63% in 30 min incubation while drying patterns at room temperature for 3 days resulted in significant peptide loss because its structure damage.

[Microencapsulated multicellular tumor spheroids: preparation and use as a novel in vitro model for drug screening].

In the current study a technique for microencapsulation of human breast adenocarcinoma cells MCF-7 in alginate-chitosan microcapsules is used. Microencapsulation is proposed to generate multicellular tumor spheroids (MTS) based on these cells and to test them further as an in vitro model for anti-tumor drug screening. Cytotoxicity of methotrexate (MTX) was studied on the obtained MTS. A set of MTS with mean size of 150, 200 and 300 m was prepared in function of a cultivation time. After incubation of MTS in cultivation medium containing MTX at concentrations of 1, 2, 10, 50 and 100 nM for 48 hs cell viability was evaluated. MTS were shown to be more resistant to MTX than the monolayer culture, and the resistance to MTX was increased with enhancing a spheroid size. At MTX concentration of 100 nM a number of viable cells in MTS with the size of 300 m was 2.5-fold bigger than that one in monolayer culture. It is suggested that the cells in microencapsulated MTS can better mimic cell behavior in a small size solid tumor than the cells in a monolayer culture. In future microencapsulated MTS can be proposed as a novel in vitro model for anticancer drug screening.

[Biodegradable polymer microparticles with entraped herbal extracts: preparation with supercritical carbon dioxide and use for tissue repair].

Biodegradable microparticles based on poly-D,L-lactide with entrapped mixture of herbal water-soluble extracts of Plantago major and Calendula officinalis were prepared. For preparation of these microparticles the previously developed method based on the usage of supercritical carbon dioxide (SC-CO2) was proposed. Microparticles were obtained by two techniques: 1) by preparing porous polymer monolith containing entrapped mixture of herbal extracts, which was then reduced to fine microparticles (ca. 0.1 mm) by dry ice grinding (called here as "monolithisation technique") and 2) by spraying of this polymer/extracts mixture through a jet (spray technique). In vitro release kinetic profile of herbal extract mixture was found to depend on the microparticle preparation technique, on the microparticle structure as well as on the initial ratio polymer/extracts (w/w). The microparticles were used for gastric ulcer treatment in a rat model. The extracts released from microparticles were found to accelerate tissue repair.

[Biodegradable microcapsules containing DNA for the new DNA vaccine design].

A general method for the preparation of biodegradable microcapsules capable of antigen inclusion is suggested. Multilayer microcapsules were obtained by the method of level-by-level sorption of various polyelectrolytes (alginate, poly-L-lysine, kappa-carrageenan, and chitosan and dextran derivatives). High inclusion efficiency was found for protein and plasmid DNA (no less than 90%). A series of microcapsules with included pTKShi plasmid that incorporated a genome site encoding the E(2) polypeptide of the classic pig plague virus were obtained for carrying out in vivo experiments. It was shown that introduction to mice of microcapsules with the included pTKShi plasmid induced an immune response. The highest antibody titers of the mouse blood sera were obtained in immunization by microcapsules based on the modified dextran/carrageenan and modified chitosan/carrageenan. The method of antigen inclusion into biodegradable microcapsules could be used for the development of encapsulated vaccines of a new generation (DNA vaccines).

[Alginate-chitosan microspheres for the specific sorption of antibodies].

Potentially hemocompatible alginate-chitiosan microparticles and microcapsules coated with a semipermeable membrane with incorporated glycoconjugates were synthesized. The membrane acts as a barrier, which keeps the incorporated glycoconjugate from going outside but permits antibodies to penetrate inside and specifically bind to antigens, high-molecular polysaccharide conjugates. The carriers obtained are highly competitive in sorption capacity with Sepharose modified by the same oligosaccharides.

[Polyelectrolyte microcapsules as systems for delivery of biologically active substances].

Novel biodegradable microcapsules for delivery of biologically active substances (BAS) were prepared by layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, namely sodium alginate (Alg) and poly-L-lysine (PLL). To immobilize these BAS, porous spherical CaCO3 microparticles were used as templates. The templates (cores) were coated with several layers of oppositely charged polyelectrolytes forming shell on a core surface. The core-shell microparticles were converted into hollow microcapsules by a core dissolution after an EDTA treatment. Mild conditions for microcapsule fabrication allow to perform an entrapment of various biomolecules while keeping their bioactivity. Biocompatibility and biodegradable capability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin (Chym) entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme was found to keep about 86% of the activity compared to a native Chym. The obtained microcapsules were stable at an acidic medium while they could be easily decomposed by trypsin treatment at an slightly alkaline medium. Chym was shown to be active after being released from the microcapsules decomposed by trypsin treatment. Thus, the microcapsules prepared by the LbL - technique can be used for the development of new type of BAS delivery systems in humans and animals.

[Entrapment of herbal extracts in biodegradable microcapsules].

The microcapsules with entrapped herbal water-soluble extracts Plantago major and Calendula officinalis L. (HE) were prepared by LbL-adsorption of carrageenan and modificated chitosan onto CaCO3 microparticles with their subsequent dissolving after the treatment of EDTA. Entrapment of HE was performed by adsorption and co-precipitation techniques. The co-precipitation provided better entrapment of HE compared to adsorption. In vitro release kinetics in an artificial gastric juice (AGJ) was studied. The HE release was shown to accelerate gastric ulcer treatment in a rat model.

[Biodegradable microparticles with immobilized peptide for wound healing].

Thrombin receptor agonist peptide (TRAP-6) may effectively replace thrombin for stimulation of damaged tissue regeneration. (Thrombin employment is limited by its high cost, instability and proinflammatory effect at high concentrations.) Immobilization of TRAP-6 into a poly(D,L)-lactide-co-glycolide (PLGA)-based matrix can protect peptides from a destruction by peptidases located in a wound area, and can also provide controlled release of the peptide. PLGA microparticles with immobilized peptide were produced by double emulsion/evaporation technique. An observation of microparticle morphology by scanning electron microscopy highlighted that peptide immobilization resulted in the increase of the microparticle porosity. TRAP-6 release kinetics was characterized by burst increase of TRAP-6 concentration in HEPES buffer solution (pH 7.5) for first 2 hours from the beginning of the experiment, and TRAP-6 complete release occurred for 20 hours. An investigation of TRAP-6 destruction by scanning electron microscopy revealed that the increase of microparticle size and surface porosity were observed already after 1 day of incubation in the buffer solution, and an aggregation of destructing microparticles was obvious by the 7th day of the incubation. Thus, peptide immobilization into PLGA microparticles can allow to develop a novel controlled release drug delivery system.

Thrombin receptor agonist Peptide immobilized in microspheres stimulates reparative processes in rats with gastric ulcer.

The effect of synthetic thrombin receptor (PAR1) agonist peptide encapsulated in microspheres made of lactic and glycolic acid copolymer on tissue reparation was studied in rats with acetate-induced ulcer. PAR1 agonist peptide was immobilized in biodegraded lactic and glycolic acid microspheres by double emulgation, the kinetics of peptide release was analyzed, and the dynamics of ulcer healing was studied in experimental (administration of microspheres with the peptide into the stomach) and two control groups (administration of saline or spheres without peptide). Thrombin receptor agonist peptide gradually released from lactic and glycolic acid microspheres into the stomach shortened the inflammation phase and shifted the proliferation phase to the earlier period, thus accelerating healing of experimental ulcers in rats.

Effect of synthetic peptide thrombin receptor agonist encapsulated in microparticles based on lactic and glycolic acid copolymer on healing of experimental skin wounds in mice.

PAR1 peptide thrombin receptor agonist (PAR1-AP) was encapsulated in microcorpuscles based on lactic and glycolic acid copolymer. The desorption profile of the preparation was studied in vitro and its wound-healing effects were studied on a model of cut skin wound in mice. The study showed that 90% PAR1-AP was desorbed over 6 h, but the peptide was detected in eluates from the microparticle surface after 23 h. The desorbed peptide retained its physiological activity and was capable of activating PAR1 receptors on human platelets. The study of the dynamics of experimental skin wound healing in mice showed lower number of macrophages in the wounds treated with PAR1-AP microparticles compared to the control (open wounds and wounds covered with microparticles) and higher number of fibroblasts on day 3 of tissue reparation. Hence, PAR1-AP desorbed from microparticles shortened the inflammation phase in the wound. On day 7 the best healing parameters were also observed in wounds treated with PAR1-AP microparticles, which attests to shortening of the proliferation phase and acceleration of wound healing.

[Polymer coatings with immobilized thrombin and peptides: preparation and use for wound healing]. / Polimernye pokrytiia s immobilizovannymi v nikh trombinom ili peptidami: poluchenie i primenenie dlia ranozazhivleniia.

Polymer dressings with encapsulated thrombin or synthetic peptides which can mimic thrombin action are employed for wound healing. Paper describes the method for preparation of these hydrogel composites of PVCL-CaAlg [poly(N-vinyl caprolactam-calcium alginate). The effect of encapsulated thrombin/peptides on tissue repair process have beet investigatat in vivo experiments using a mouse model of wound healing. The developed dressings accelerated wound healing: thascan be used as a basis for creation of novel formulations with controlled drug release for wound therapy.

Immobilized thrombin receptor agonist peptide accelerates wound healing in mice.

To accelerate the healing processes in wound repair, attempts have been repeatedly made to use growth factors including thrombin and its peptide fragments. Unfortunately, the employment of thrombin is limited because of its high liability and pro-inflammatory actions at high concentrations. Some cellular effects of thrombin in wound healing are mediated by the activation of protease activated receptor-1 (PAR-1). The thrombin receptor agonist peptide (TRAP:SFLLRN) activates this receptor and mimics the effects of thrombin, but TRAP is a relatively weak agonist. We speculated that the encapsulated peptide may be more effective for PAR-1 activation than nonimmobilized peptide and developed a novel method for TRAP encapsulation in hydrogel films based on natural and synthetic polymers. The effects of an encapsulated TRAP in composite poly(N-vinyl caprolactam)-calcium alginate (PVCL) hydrogel films were investigated in a mouse model of wound healing. On day 7 the wound sizes decreased by about 60% under TRAP-chitosan-containing PVCL films, as compared with control films without TRAP. In the case of TRAP-polylysine-containing films no significant decrease in wound sizes was found. The fibroblast/macrophage ratio increased under TRAP-containing films on day 3 and on day 7. The number of proliferating fibroblasts increased to 150% under TRAP-chitosan films on day 7 as compared with control films. The number of [3H]-thymidine labeled endothelial and epithelial cells in granulation tissues was also enhanced. Thus, the immobilized TRAP to PVCL-chitosan hydrogel films were found to promote wound healing following the stimulation of fibroblast and epithelial cell proliferation and neovascularization. Furthermore, TRAP was shown to inhibit the secretion of the inflammatory mediator PAF from stimulated rat peritoneal mast cells due to augmentation of NO release from the mast cells. The encapsulated TRAP is suggested to accelerate wound healing due to the anti-inflammatory effects and earlier development of the proliferative phase of wound healing.

[Isolation, crystallization, and preliminary x-ray study of a Fab fragment of monoclonal antibody against human interleukin-2 and its complex with antigenic peptide]. / Poluchenie, kristallizatsiia i predvaritel'nye rentgenostrukturnye issledovaniia Fab-fragmenta monoklonal'nogo antitela k interleikinu-2 cheloveka i ego kompleksa s antigennym peptidom.

Antigen-binding fragments (Fab) of mouse monoclonal antibodies to human interleukin-2 were obtained in preparative quantities by a modified procedure. These Fab-fragments were shown to be homogeneous according to the isoelectric focusing method. Various monocrystals of these free Fab-fragments and their complexes with the antigenic peptide corresponding to the 59-72 sequence of interleukin-2 were obtained. These were shown to be suitable for X-ray and were preliminarily studied by X-ray.

[Molecular design of polymeric materials for biotechnology and medicine]. / Molekuliarnoe konstruirovanie polimernykh materialov dlia biotekhnologii i meditsiny.

This review describes new polymer materials for biomedical applications developed in the Polymers for Biology Laboratory of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences. These include composite rigid sorbents for biochromatography, polymer dispersions for immunoassay, polymer hydrogels for immobilization of enzymes and cells, and polymer ultra thin films as biomembrane models and materials for biosensors. Some general and specific properties of these new materials and models as well as examples of their applications are discussed.

[Thrombin--a regulator of reparative processes in wound healing]. / Trombin--reguliator reparativnikh protsessov pri zazhivlenii ran.

Thrombin, binding to receptors of the protease activated receptor (PAR) family, is involved in wound healing by inducing the reparation processes and regulating the activity of mast cells, which secrete mediators of inflammation. Using thrombin receptor agonist peptide (TRAP-6) for the activation of rat mast cells, effect of several receptors, including PAR-1, on mast cells was demonstrated. It was shown that TRAP increases the concentration of Ca2+ in the cytoplasm of mast cells and regulates cell degranulation, while releasing nitrogen oxide. Thrombin encapsulated in poly(N-vinyl caprolactam)-calcium alginate (PVCL-Ca-Alg) hydrogel films promotes wound healing in rats as demonstrated by the acceleration of fibroblast proliferation and neovascularization.

Stabilization of proteases by entrapment in a new composite hydrogel.

A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caprolactam) hydrogel was developed that provided 75-90% retention of the activity of entrapped enzymes compared to soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25 degrees C higher than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various reactions proceeding within wide pH and temperature ranges.